Increasing cancer patient survival time by administration of dithio-containing compounds

ABSTRACT

The present invention discloses and claims compositions, methods of treatment, and kits which cause an increase in the time of survival in cancer patients, wherein the cancer: (i) overexpresses thioredoxin or glutaredoxin and/or (ii) exhibits evidence of thioredoxin- or glutaredoxin-mediated resistance to one or more chemotherapeutic interventions. The present invention also discloses and claims methods and kits for the administration of said compositions to properly treat cancer patients. Additionally, the present invention discloses and claims methods and kits for quantitatively determining the level of expression of thioredoxin or glutaredoxin in the cancer cells of a cancer patient, methods of using those determined levels in the initial diagnosis and/or planning of subsequent treatment methodologies for said cancer patient, as well as ascertaining the potential growth “aggressiveness” of the particular cancer and treatment responsiveness of the particular type of cancer. Further, the present invention discloses and claims novel pharmaceutical compositions, methods, and kits used for the treatment of patients with medical conditions and disease where there is the overexpression of thioredoxin and/or glutaredoxin, and wherein this overexpression is associated with deleterious physiological effects in the patients.

The present invention relates to novel pharmaceutical compositions,methods, and kits used for the treatment of cancer and other medicalconditions. More specifically, the present invention relates to novelpharmaceutical compositions, methods, and kits comprising medicamentsused for the treatment of lung cancer, adenocarcinoma, and other medicalconditions. In addition, the present invention also relates tocompositions, methods of treatment, and kits which cause an increase intime of survival in cancer patients, wherein the cancer either: (i)overexpresses thioredoxin or glutaredoxin and/or (ii) exhibits evidenceof thioredoxin- or glutaredoxin-mediated resistance to one or morechemotherapeutic interventions. The present invention also relates tomethods and kits for the administration of said compositions to properlytreat cancer patients. Additionally, the present invention relates tomethods and kits for quantitatively determining the level of expressionof thioredoxin or glutaredoxin in the cancer cells of a cancer patient,methods of using those determined levels in the initial diagnosis and/orplanning of subsequent treatment methodologies for said cancer patient,as well as ascertaining the potential growth “aggressiveness” of theparticular cancer and treatment responsiveness of the particular type ofcancer. Further, the present invention relates to novel pharmaceuticalcompositions, methods, and kits used for the treatment of patients withmedical conditions and diseases where there is the overexpression ofthioredoxin and/or glutaredoxin, and wherein this overexpression isassociated with deleterious physiological effects in the patients.

BACKGROUND OF THE INVENTION

As the number of agents and treatment regimens for cancer has increased,clinicians and researchers are seeking to fully elucidate thebiological, chemical, pharmacological, and cellular mechanisms which areresponsible for the pathogenesis and pathophysiology of the variousadverse disease manifestations, as well as how chemotherapeutic drugsexert their anti-cancer and cytotoxic or cytostatic activity on abiochemical and pharmacological basis. As described herein, with theexception of the novel conception and practice of the present invention,there are no currently-approved compositions which markedly increase thesurvival time of a cancer patient via a targeted therapeutic interactionthat involves the direct modulation of either the thioredoxin orglutaredoxin pathways, thereby leading to increased anti-cancer andcytotoxic effects of the chemotherapeutic agent(s) within the cancercells. Moreover, prior to the clinical studies described in the presentinvention, no clinical studies utilizing the novel treatment methods andcompositions disclosed herein have observed “an increase in patientsurvival time” in a medically-important manner, but rather measured only“patient response” (i.e., tumor response—a shrinkage of tumor that isobserved radiographically). These are highly innovative and novelfeatures of the present invention.

It has been increasingly recognized that many different types of cancercells have been shown to have increased expression and/or activity ofthioredoxin and/or glutaredoxin including, but not limited to, lungcancer, colorectal cancer, gastric cancer, esophageal cancer, ovariancancer, cancer of the biliary tract, gallbladder cancer, cervicalcancer, breast cancer, endometrial cancer, vaginal cancer, prostatecancer, uterine cancer, hepatic cancer, pancreatic cancer, andadenocarcinoma.

Thioredoxin and glutaredoxin are members of the thioredoxin superfamily;that mediate disulfide exchange via their Cys-containing catalyticsites. While glutaredoxins mostly reduce mixed disulfides containingglutathione, thioredoxins are involved in the maintenance of proteinsulfhydryls in their reduced state via disulfide bond reduction. See,e.g., Print, W. A., et al., The role of the thioredoxin and glutaredoxinpathways in reducing protein disulfide bonds in the Escherichia colicytoplasm. J. Biol. Chem. 272:15661-15667 (1996). The reduced form ofthioredoxin is generated by the action of thioredoxin reductase; whereasglutathione provides directly the reducing potential for regeneration ofthe reduced form of glutaredoxin. Glutaredoxins are oxidized bysubstrates, and reduced non-enzymatically by glutathione. In contrast tothioredoxins, which are reduced by thioredoxin reductase, nooxidoreductase or substrate, other than those described in the presentinvention, has been reported to specifically reduce glutaredoxins.Instead, oxidized glutathione is regenerated by glutathione reductase.Together these components comprise the glutathione system. See, e.g.,Holmgren, A. and Fernandes, A. P., Glutaredoxins: glutathione-dependentredox enzymes with functions far beyond a simple thioredoxin backupsystem. Antioxid. Redox. Signal. 6:63-74 (2004); Holmgren, A.,Thioredoxin and glutaredoxin systems. J. Biol. Chem. 264:13963-13966(1989). The thioredoxin system, together with the glutathione system, isregarded as a main regulator of oxidative metabolism involving theintracellular redox environment, exercising control of the cellularredox state and antioxidant defense, as well as governing the redoxregulation of several cellular processes. The system is involved indirect regulation of (i) several transcription factors, (ii) apoptosis(i.e., programmed cell death) induction, and (iii) many metabolicpathways (e.g., DNA synthesis, glucose metabolism, selenium metabolism,and vitamin C recycling). See, e.g., Amér, E. S. J., et al.,Physiological functions of thioredoxin and thioredoxin reductase. Eur.J. Biochem. 267:6102-6109 (2000).

In brief, the overexpression (or increased activity, or both) ofthioredoxin or glutaredoxin in cancer cells mediates a multi-componentand multi-pathway mechanism which confers a survival advantage to cancercells. Overexpression/increased levels or responsiveness mediated bythioredoxin and/or glutaredoxin in cancer cells can lead to severalimportant biological alterations including, but not limited to: (i) lossof apoptotic sensitivity to therapy (i.e., drug or ionizing radiationresistance); (ii) increased conversion of RNA into DNA (involvingribonucleotide reductase); (iii) altered gene expression; (iv) increasedcellular proliferation signals and rates; (v) increased thioredoxinperoxidase; and (vi) increased angiogenic activity (i.e., increasedblood supply to the tumor). Accordingly, by pharmacological inactivationor modulation of thioredoxin and/or glutaredoxin by the proper medicaladministration of effective levels and schedules of the compositions ofthe present invention, can result in enhancement of chemotherapy effectsand thereby lead to increased patient survival.

The compositions of the present invention comprise amedically-sufficient dose of an oxidative metabolism-affecting Formula(I) compound. The compounds of Formula (I) includepharmaceutically-acceptable salts of such compounds, as well asprodrugs, analogs, conjugates, hydrates, solvates and polymorphs, aswell as stereoisomers (including diastereoisomers and enantiomers) andtautomers of such compounds. The Formula (I) compounds of the presentinvention also comprise a medically-sufficient dose of the disodium saltof 2,2′-dithio-bis-ethane sulfonate, which has been referred to in theliterature as Tavocept™, dimesna, and BNP7787. The compositions of thepresent invention also comprise a medically-sufficient dose of themetabolite of disodium 2,2′-dithio-bis-ethane sulfonate, known as2-mercapto ethane sulfonate sodium (also known in the literature asmesna) and 2-mercapto ethane sulfonate as a disulfide form which isconjugated with a substituent group consisting of:

-Cys, -Homocysteine, -Cys-Gly, -Cys-Glu, -Homocysteine,-Homocysteine-Gly, -Homocysteine-Glu, -Cys-Glu,

wherein R₁ and R₂ are any L- or D-amino acids.

The underlying mechanisms of the Formula (I) compounds of the presentinvention in increasing the survival time of cancer patients involvesone or more of several novel pharmacological and physiological factors,including but not limited to, a prevention, compromise and/or reductionin the levels, responsiveness, or in the concentration and/or tumorprotective metabolism of various physiological cellular thiols; theseantioxidants and enzymes are increased in concentration and/or activityin cancer cells, respectively, due in part to activation and/oroverexpression of thioredoxin and/or glutaredoxin levels or activitywhich are present in many cancer cells, and this increase inconcentration and/or activity may be may be further enhanced by exposureto cytotoxic chemotherapeutic agents in tumor cells. The Formula (I)compounds of the present invention may exert therapeutic medicinal andpharmacological activity by the intrinsic composition of the moleculeitself (i.e., an oxidized disulfide), as well as by oxidizing freethiols to form oxidized disulfides (i.e., by non-enzymatic SN2-mediatedreactions, wherein attack of a thiol/thiolate upon a disulfide leads tothe scission of the former disulfide which is accompanied by the faciledeparture of a thiol-containing group). As the thiolate group is farmore nucleophilic than the corresponding thiol, the attack is believedto be via the thiolate, however, in some cases the sulfur atom containedwithin an attacking free sulfhydryl group may be the nucleophile), andmay thereby lead to pharmacological depletion and metabolism ofreductive physiological free thiols (e.g., glutathione, cysteine, andhomocysteine).

Overexpression/increased levels or increased responsiveness mediated bythioredoxin and/or glutaredoxin in cancer cells leads to loss ofapoptotic sensitivity to therapy (i.e., drug or ionizing radiationresistance), increased conversion of RNA into DNA (involvingribonucleotide reductase), increased gene expression, increasedthioredoxin peroxidase, and increased angiogenic activity (i.e.,increased blood supply to the tumor). Accordingly, pharmacologicalinactivation or modulation of thioredoxin and/or glutaredoxin by theproper medical administration of effective levels and schedules of thecompositions of the present invention can result in increased patientsurvival.

It is believed by the Applicant of the present invention that theseaforementioned mechanisms of action are mediated by the Formula (I)compounds of the present invention and metabolites thereof (e.g.,2-mercapto ethane sulfonate (mesna) and mesna heteroconjugates) and aredirectly involved in the marked increase in the survival time ofpatients suffering from cancer including, but not limited to, non-smallcell lung carcinoma (NSCLC) or adenocarcinoma who received treatmentsutilizing the compositions, formulation, and methods of the presentinvention. This has extremely important implications for advancing thetreatment of patients with cancer.

Compositions and formulations comprising the Formula (I) compounds ofthe present invention may be given using any combination of thefollowing three general treatment methods: (i) in a direct inhibitory orinactivating manner (i.e., direct chemical interactions that inactivatethioredoxin and/or glutaredoxin) and/or depletive manner (i.e.,decreasing thioredoxin and/or glutaredoxin concentrations or productionrates), thereby increasing the susceptibility of the cancer cells to anysubsequent administration of any chemotherapeutic agent or agents thatmay act directly or indirectly through the thioredoxin- and/orglutaredoxin-mediated pathways in order to sensitize the patient'scancer and thus increase the survival of the patient; and/or (ii) in asynergistic manner, where the anti-thioredoxin and/or glutaredoxintherapy is concurrently administered with chemotherapy administrationwhen a cancer patient begins any chemotherapy cycle, in order toincrease and optimize the pharmacological activity directed againstthioredoxin- and/or glutaredoxin-mediated mechanisms present whilechemotherapy is being concurrently administered; and/or (iii) in apost-treatment manner (i.e., after the completion of chemotherapy doseadministration or a chemotherapy cycle) in order to maintain thepresence of a pharmacologically-induced depletion, inactivation, ormodulation of thioredoxin and/or glutaredoxin in the patient's cancercells for as long as optimally required. Additionally, theaforementioned compositions and formulations may be given in anidentical manner to increase patient survival time in a patientreceiving treatment with a cytotoxic or cytostatic anti-cancer agent byany additionally clinically-beneficial mechanism(s).

I. Oxidative Metabolism

In its most simple terms, oxidative metabolism refers to the enzymaticpathways leading to the addition of oxygen (i.e., oxidation) or theremoval of electrons or hydrogen (i.e., reduction) from intermediates inthe pathways. The redox state of any particular biological environmentcan be defined as the sum of oxidative and reductive processes occurringwithin that environment which, in turn, directly relates to the extentto which molecules are oxidized or reduced within it. The redoxpotential of biological ions or molecules is a measure of their tendencyto lose an electron (i.e., thereby becoming oxidized) and is expressedas E₀ in volts. The more strongly reducing an ion or molecule, the morenegative its E₀. As previously stated, under normal physiologicalcircumstances, most intracellular biological systems are predominantlyfound in a reduced state. Within cells, thiols (R—SH) such asglutathione (GSH), cysteine, homocysteine, and the like, are maintainedin their reduced state, as are the nicotinamide nucleotide coenzymesNADH and NADPH. The opposite relationship is found in plasma, where thehigh partial pressure of oxygen (pO₂) promotes an oxidative environment,thereby leading to a high proportion (i.e., greater than 90%) of thephysiological sulfur-containing amino acids and peptides (e.g.,glutathione (GSH)) existing in stable oxidized (disulfide) forms. Inplasma, there are currently no known enzymes that appear to reduce thedisulfide forms of these sulfur-containing amino acids and GSH; thisfurther contributes to the plasma vs. cellular disparity in terms of therelative proportions of physiological disulfides vs. thiols.Physiological circumstances can, however, arise which alter the overallredox balance and lead to a more oxidizing environment in the cell.Various complex physiological systems have evolved to remove, repair,and control the normal reducing environment. However, when the oxidizingenvironment overwhelms these protective mechanisms, oxidative damage andprofound biological and toxic activity can occur.

In biological systems, the formation of potentiallyphysiologically-deleterious reactive oxygen species (ROS) and that ofreactive nitrogen species (RNS), may be caused from a variety ofmetabolic and/or environmental processes. By way of non-limitingexample, intracellular ROS (e.g., hydrogen peroxide: H₂O₂; superoxideanion: O₂ ⁻; hydroxyl radical: OH⁻; nitric oxide: NO; and the like) maybe generated by several mechanisms: (i) by the activity of radiation,both exciting (e.g., UV-rays) and ionizing (e.g., X-rays); (ii) duringxenobiotic and drug metabolism; and (iii) under relative hypoxic,ischemic and catabolic metabolic conditions, as well as by exposure tohyperbaric oxygen. Protection against the harmful physiological activityof ROS and RNS species is mediated by a complex network of overlappingmechanisms and metabolic pathways that utilize a combination of smallredox-active molecules and enzymes coupled with the expenditure ofreducing equivalents. These complex networks of mechanisms, metabolicpathways, small redox-active molecules, and enzymes will be fullydiscussed, infra.

Concentrations of ROS and RNS which cannot be adequately dealt with bythe endogenous antioxidant system can lead to damage of lipids,proteins, carbohydrates, and nucleic acids. Changes in oxidativemetabolism which lead to an increase in the oxidizing environment andthe formation of potentially physiologically-deleterious reactive oxygenspecies (ROS) and that of reactive nitrogen species (RNS) has beengenerally termed within the literature as “oxidative stress”. It hasalso recently been recognized that cancer cells may respond to such“oxidative stress”, induced by chemotherapy or radiation exposure, bydecreasing the concentrations of ROS and oxidized thiols and well as byincreased concentrations of thiol and anti-oxidants. It should be notedthat when either or both of these mechanisms are operative, thesubject's tumor cells may become resistant to chemotherapy and radiationtherapy, thereby representing an important obstacle to curing orcontrolling the progression of the subject's cancer.

The putative mechanisms of the Formula (I) compositions of the presentinvention which function in the potentiation of the anti-cancer activityof chemotherapeutic agents may involve one or more of several novelpharmacological and physiological factors, including but not limited to,a prevention, compromise, and/or reduction in the normal increase,responsiveness, or in the concentration and/or tumor protectivemetabolism of glutathione/cysteine and other physiological cellularthiols; these antioxidants and enzymes are increased in concentrationand/or activity, respectively, in response to the induction ofintracellular oxidative stress which may be caused by exposure tocytotoxic chemotherapeutic agents in tumor cells. Additional informationregarding certain mechanisms which may be involved in the biologicalactivities of the Formula (I) compounds is disclosed in U.S. patentapplication Ser. No. 11/724,933, filed Mar. 16, 2007, the disclosure ofwhich is hereby incorporated by reference in its entirety.

II. Physiological Cellular Thiols

Thiol groups are those which contain functional CH₂—SH groups withinconserved cysteinyl residues. It is these thiol-containing proteinswhich have been elucidated to play the primary role in redox-sensitivereactions. Their redox-sensing abilities are thought to occur byelectron flow through the sulfhydryl side-chain. Thus, it is the uniqueproperties afforded by the sulfur-based chemistry in protein cysteines(in some cases, possibly in conjunction with chelated central metalatoms) that is exploited by transcription factors which “switch” betweenan inactive and active state in response to elevated concentrations ofROS and/or RNS. It should be noted that the majority of cellular proteinthiols are compartmentalized within highly reducing environments and aretherefore “protected” from such oxidation. Hence, only proteins withaccessible thiol moieties, and higher oxidation potentials are likely tobe involved in redox-sensitive signaling mechanisms.

There are numerous naturally-occurring thiols and disulfides that areinvolved in oxidative metabolism. The most abundantbiologically-occurring amino acid is cysteine, along with its disulfideform, cystine. Another important and highly abundant intracellular thiolis glutathione (GSH), which is a tripeptide comprised ofγ-glutamate-cysteine-glycine. Thiols can also be formed in those aminoacids which contain cysteine residues including, but not limited to,cystathionine, taurine, and homocysteine. Many oxidoreductases andtransferases rely upon cysteine residues for their physiologicalcatalytic functions. There are also a large number of low molecularweight cysteine-containing compounds, such a Co-enzyme A andglutathione, which are vital enzymes in maintaining oxidative/reductivehomeostasis in cellular metabolism. These compounds may also beclassified as non-protein sulfhydryls (NPSH).

Structural and biochemical data has also demonstrated thatthiol-containing cysteine residues and the disulfide cystine, play aubiquitous role in allowing proteins to respond to ROS. Theredox-sensitivity of specific cysteine residues imparts specificity toROS-mediated cellular signaling. By reacting with ROS, cysteine residuesfunction as “detectors” of redox status; whereas the consequent chemicalchange in the oxidized cysteine can be converted into a proteinconformational change, hence providing an activity or response.

Within biological systems, thiols undergo a reversibleoxidation/reduction reaction, as illustrated below, which are oftencatalyzed by transition metals. These reactions can also involve freeradicals (e.g., thioyl RS) as intermediates. In addition, proteins whichpossess SH/SS groups can interact with the reduced form of GSH in athiol-disulfide exchange. Thiols and their disulfides are reversiblylinked, via specific enzymes, to the oxidation and reduction of NADP andNADPH. This reversible oxidation/reduction reaction is shown in Table 1,below:

TABLE 1

There is increasing experimental evidence that indicates thatthiol-containing proteins are sensitive to thiol modification andoxidation when exposed to changes in the redox state. This sensing ofthe redox potential is thought to occur in a wide range of diversesignal transduction pathways. Moreover, these redox sensing proteinsplay roles in mediating cellular responses to changes in intracellularoxidative metabolism (e.g., increased cellular proliferation).

One of the primary enzymes involved in the synthesis of cellular thiolsis cysteine synthase, which is widely distributed in human tissues,where it catalyzes the synthesis of cysteine from serine. The absorptionof cystine and structurally-related amino acids (e.g., ornithine,arginine, and lysine) are mediated by a complex transporter system. TheXc transporter, as well as other enzymes, participate in these cellularuptake mechanisms. Once transported into the cell, cystine is rapidlyreduced to cysteine, in an enzymatic reaction which utilizes reducedglutathione (GSH). In the extracellular environment, the concentrationsof cystine are typically substantially higher than cysteine, and whereasthe reverse is true in the intracellular environment.

III. Lung Cancer

Lung cancer is reported to be the leading cause of smoking- andcancer-related mortality in both sexes. The prevalence of lung cancer issecond only to that of prostate cancer in men and breast cancer inwomen. In the United States, lung cancer was reported recently tosurpass heart disease as the leading cause of smoking-related mortality.Most lung carcinomas are diagnosed at an advanced stage, conferring apoorer prognosis. Lung cancer is estimated to be the cause of 921,000deaths each year worldwide, accounting for approximately 18% of allcancer-related deaths. Lung cancer is highly lethal, with a 5-yearpatient survival rate of only 14% being observed in the United States.An estimated 164,100 (i.e., 89,500 in men and 74,600 in women) new lungcancer cases will occur this year (2008) in the United States. See,e.g., National Cancer Institute-2008 Lung Cancer Estimates(www.Cancer.gov).

Lung cancer manifests with symptoms produced by the primary tumor,locoregional spread, metastatic disease, or ectopic hormone production.Approximately 7-10% of patients with lung cancer are asymptomatic andtheir cancers are diagnosed incidentally after a chest x-ray performedfor other reasons. The symptoms produced by the primary tumor depend onits location (e.g., central, peripheral).

Of the symptoms produced by the primary tumor, central tumors aregenerally squamous cell carcinomas and produce symptoms or signs ofcough, dyspnea, atelectasis, post-obstructive pneumonia, wheezing, andhemoptysis, and peripheral tumors are generally adenocarcinomas or largecell carcinomas and, in addition to causing cough and dyspnea, can causesymptoms or signs from pleural effusion and severe pain as a result ofinfiltration of parietal pleura and the chest wall. Symptoms due tolocoregional spread can include: (i) superior vena cava obstruction;(ii) paralysis of the left recurrent laryngeal nerve and phrenic nervepalsy (causing hoarseness and paralysis of the diaphragm); (iii)pressure on the cervical sympathetic plexus (causing Horner syndrome);(iv) dysphagia resulting from esophageal compression; (v) pericardialeffusion and cardiac tamponade; and (vi) superior sulcus apical primarytumors can cause compression of the brachial plexus roots as they exitthe neural foramina, causing intense, radiating neuropathic pain in theipsilateral upper extremity (e.g., Pancoast tumors). Lung cancer isassociated with a variety of paraneoplastic syndromes: (i) most of suchparaneoplastic syndromes are associated with small cell lung cancer;(ii) squamous cell carcinomas are more likely to be associated withhypercalcemia due to parathyroidlike hormone production; and (iii)clubbing and hypertrophic pulmonary osteoarthropathy and the Trousseausyndrome of hypercoagulability are caused more frequently byadenocarcinomas. Eaton-Lambert myasthenic syndrome is reported inassociation with small cell and non-small cell lung cancers.Paraneoplastic syndromes can pose debilitating problems in cancerpatients and can complicate the medical management of such patients.

Non-small cell lung cancer (NSCLC) accounts for more than 80% of allprimary lung cancer, and surgically resectable (with curative intent)cases account for less than 30%. Chemotherapy and radiotherapy are themainstays of treatment in unresectable cases, but the median survivalperiod is only 15-20 months and the 3-year survival rate isapproximately 30-40% in stage IIIA and IIIB cases. The prognosis is evenworse in stage IV patients with a median survival period of 8-10 monthsand a 1-year survival rate of less than 30%. At these advanced stages,the main therapeutic objectives are increasing the survival period andpreserving the quality of life; these patients are not generallyconsidered curable. It is important to consider the important concept ofincreasing the observed survival rate as a prerequisite for achieving acurative outcome in any therapeutic intervention that involves a definedpatient population (e.g., non-small cell lung cancer patients) that isconsidered to be incurable. See, e.g., Cortes-Funes H., New TreatmentApproaches for Lung Cancer and Impact on Survival. Semin. Oncol.29:26-29 (2002); Fukuoka, M and Saijoh, N., Practical medicine—Lungcancer, Nannkodo (2001). NSCLC is pathologically characterized furtherinto adenocarcinoma, squamous cell carcinoma, large cell carcinoma, andother less common forms. Clinically there are also important differencesin NSCLC that can be observed in smokers and non-smokers.

A summary of clinical characteristics by histologic NSCLC subtypeinclude:

-   -   Adenocarcinoma is the most frequent non-small cell lung cancer        (NSCLC) in the United States, representing 35% to more than 50%        of all lung cancers, usually occurring in a peripheral location        within the lung and arising from bronchial mucosal glands.        Adenocarcinoma is the most common histologic subtype,        manifesting as a scar carcinoma. This is a subtype observed most        commonly in persons who do not smoke, however, adenocarcinoma is        also common in smokers. This type of NSCLC may also manifest as        multifocal tumors in a bronchoalveolar form. Bronchoalveolar        carcinoma is a distinct subtype of adenocarcinoma with the        classic manifestation as an interstitial lung disease upon        radiographic imaging. Bronchoalveolar carcinoma arises from type        II pneumocytes and grows along alveolar septa. This subtype may        manifest as a solitary peripheral nodule, multifocal disease, or        a rapidly progressing pneumonic form. A characteristic finding        in persons with advanced disease is voluminous watery sputum.        Overexpression of thioredoxin and/or glutaredoxin has been noted        in adenocarcinomas of the lung.    -   Squamous cell carcinoma accounts for approximately 25-30% of all        lung cancers. The classic manifestation is a cavitary lesion in        a proximal bronchus. This type is characterized histologically        by the presence of keratin pearls and can be detected based on        results from cytologic studies because it has a tendency to        exfoliate. It is the type most often associated with        hypercalcemia.    -   Large cell carcinoma accounts for approximately 10-15% of lung        cancers, typically manifesting as a large peripheral mass upon        radiographic imaging. Histologically, this type has sheets of        highly atypical cells with focal necrosis, with no evidence of        keratinization (typical of squamous cell carcinoma) or gland        formation (typical of adenocarcinomas). Patients with large cell        carcinoma are more likely to develop gynecomastia and        galactorrhea as paraneoplastic syndromes.

Various types of lung cancer have been shown to have an increasedoxidative metabolism and/or increased concentrations of thioredoxinand/or glutaredoxin, and may further overexpress these in response tochemotherapy, thus resulting in tumor-mediated drug resistance tochemotherapy. Therefore, any tumors that possess the characteristics ofan increased oxidative metabolism and/or increased concentration ofthioredoxin and/or glutaredoxin are more amenable to the therapeuticbenefits, including increased survival outcomes that would be mediatedby an intervention from a composition or method of the presentinvention.

IV. Adenocarcinoma

Adenocarcinoma is a histopathological description and classification ofcancers that originate primarily from glandular tissue. Glandular tissuecomprises organs that synthesize a substance for release such mucin orhormones. Glands can be divided into two general groups: (i) endocrineglands—glands that secrete their product directly onto a surface ratherthan through a duct, often into the blood stream and (ii) exocrineglands—glands that secrete their products via a duct, often intocavities inside the body or its outer surface. Exocrine glands may befurther differentiated into three categories: apocrine, holocrine, andmerocrine. However, it should be noted that to be classified asadenocarcinoma, the cells do not necessarily need to be part of a gland,as long as they have secretory properties. Adenocarcinoma may be derivedfrom various tissues including, but not limited to, breast, colon, lung,prostate, salivary gland, esophagus, stomach, liver, gall bladder andbile ducts, pancreas (99% of pancreatic cancers are ductaladenocarcinomas), cervix, vagina, ovary, and uterus, prostate, as wellas unknown primary adenocarcinomas, which are not uncommon.

Adenocarcinoma is a neoplasm which frequently presents marked difficultyin differentiating from where and from which type of glandular tissuethe tumor(s) arose. Thus, an adenocarcinoma identified in the lung mayhave had its origins (or may have metastasized) from an ovarianadenocarcinoma. Cancer for which a primary site cannot be found iscalled cancer of unknown primary, and adenocarcinomas of unknown primaryare the most common type of unknown primary cancers. The primary site isidentified in only approximately 10-20% of patients during theirremaining life times and it frequently is not identified untilpost-mortem examination. It has been reported that approximately 60% ofpatients (i.e., over 50,000 patients per annum in the United States) whoare diagnosed with carcinoma of unknown primary site suffer fromadenocarcinoma.

A diagnosis of adenocarcinoma which is not further described (i.e.,adenocarcinoma not otherwise specified; adenocarcinoma NOS) is often apreliminary diagnosis and can frequently be clarified with the use ofimmunohistochemistry or fluorescent in situ hybridization (FISH) (see,e.g., Dabbs, D. J. and Silverman, J. F., Immunohistochemical andFluorescent in situ Hybridization Workup of Metastatic Carcinoma ofUnknown Primary. Path. Case Rev. 6(4):146-153 (2005)), and/or variousimaging methodologies including, but not limited to, computerizedtomography (CT), magnetic resonance imaging (MRI), and positron emissiontomography (PET).

Immunohistochemistry refers to the process of localizing proteins incells of a tissue section exploiting the principle of antibodies bindingspecifically to antigens in biological tissues. Immunohistochemistry isalso widely used in basic research to understand the distribution andlocalization of biomarkers in different parts of a tissue.Immunohistochemical staining is a widely used specialized technique inthe diagnosis of cancer and the classification of neoplasms. Theantibodies utilized may be either polyclonal or monoclonal in nature andmay be directed against cell components or products which can include:(i) enzymes (e.g., prostatic acid phosphatase, neuron-specificenoenzymes); (ii) normal tissue components (e.g., keratin,neurofilaments); and (iii) hormones or hormone receptors (e.g., estrogenreceptor, oncofetal antigens, S-100 proteins). It should be noted thatspecific molecular markers are characteristic of particular cancertypes. For example, adeno'carcinoma often gives positiveimmunohistochemical results for thyroid transcription factor-1 (TTF-1).Visualizing an antibody-antigen interaction can be accomplished in anumber of ways. In the most common instance, an antibody is conjugatedto an enzyme, such as peroxidase, that can catalyze a color-producingreaction, as with immunoperoxidase staining. Alternatively, the antibodycan also be tagged to a fluorophore, such as FITC, rhodamine, Texas Red,or DyLight Fluor, as with immunofluorescence.

Fluorescent in situ hybridization (FISH) is a cytogenetic technique thatcan be used to detect and localize the presence or absence of specificDNA sequences on chromosomes. It utilizes fluorescent-tagged nucleicacid probes that bind to only those parts of the chromosome with whichthey show a high degree of nucleotide sequence complementarily.Fluorescence microscopy can be used to find out where the fluorescentprobe bound to the chromosome.

Adenocarcinomas are quite common and arise in a variety of sites.Similar to NSCLC, it has also been shown that adenocarcinomas have anincreased oxidative metabolism and/or increased concentrations ofthioredoxin and/or glutaredoxin, and may further overexpress these inresponse to chemotherapy, resulting in tumor-mediated drug resistance tochemotherapy.

As set forth above, non-small cell lung carcinoma (NSCLC) andadenocarcinoma are highly prevalent forms of cancer and account for alarge percentage of the deaths associated with cancer world-wide. Giventhe relatively refractory nature of NSCLC and adenocarcinoma to manyforms of therapy, there remains a need for the development ofcompositions and treatment regimens that are both generally safe andeffective for increasing the survival time of patients receivingchemotherapy, slowing the progression of their tumors, and/orstimulating or maintaining the beneficial physiological function ofimportant bodily processes in normal (i.e., non-cancerous) cells andtissues. It has also been recognized that both NSCLC and adenocarcinomashave an increased oxidative metabolism and/or increased concentrationsof thioredoxin and/or glutaredoxin, and may further overexpress these inresponse to chemotherapy, resulting in tumor-mediated drug resistance tochemotherapy. Therefore, any tumors that possess these characteristicsare more amenable to the therapeutic benefits, including increasedsurvival outcomes, which would be mediated by an intervention from acomposition or method of the present invention. Recent, surprising andmedically-important new finding and functions, based upon recentclinical trial results, have been observed involving the Formula (I)compounds set forth in the present invention. These observations haveextremely important implications for the treatment of cancer and variousother medical conditions.

In addition to the foregoing considerations regarding cancer, manypatients, including cancer patients receiving chemotherapy, are also inneed of: maintaining or stimulating hematological function; maintainingor stimulating erythropoietin function or synthesis; mitigating orpreventing anemia; and maintaining or stimulating pluripotent,multipotent, and unipotent normal stem cell function or synthesis.

SUMMARY OF THE INVENTION

The invention described and claimed herein has many attributes andembodiments including, but not limited to, those set forth or describedor referenced in this Summary section. However, it should be noted thatthis Summary is not intended to be all-inclusive, nor is the inventiondescribed and claimed herein limited to, or by, the features orembodiments identified in said Summary. Moreover, this Summary isincluded for purposes of illustration only, and not restriction.

As previously discussed, many types of cancer cells have been shown tohave increased expression and/or activity of thioredoxin or glutaredoxinincluding, but not limited to, lung cancer, colorectal cancer, gastriccancer, esophageal cancer, ovarian cancer, cancer of the biliary tract,gallbladder cancer, cervical cancer, breast cancer, endometrial cancer,vaginal cancer, prostate cancer, uterine cancer, hepatic cancer,pancreatic cancer, and adenocarcinoma. The overexpression (or increasedactivity, or both) of thioredoxin and/or glutaredoxin in cancer cellsmediates a multi-component and multi-pathway survival advantage tocancer cells which becomes manifest as chemotherapy drug resistance toapoptosis. Such overexpression of either of these key oxidoreductasepathways thereby results in the lack or impediment of the intendedtherapeutic effects of medical interventions on cancer cells, andfurther results in an observed shortened patient survival that isbelieved to be mediated by the presence and persistence of increasedconcentrations or expression of thioredoxin or glutaredoxin, which inturn promote tumor-mediated resistance to chemotherapy-inducedapoptosis, overexpression of oxidoperoxidases, increased conversion ofRNA into DNA, increased nuclear transcription, increased cellproliferation, and/or increased angiogenesis, any of which can act inconcert to provide the cancer cells the ability to resist the cytotoxicactions of chemotherapy and radiation therapy and thereby decrease thetime of patient survival.

The present invention involves the medicinal and pharmacologicalinactivation and modulation of the thioredoxin/glutaredoxin system whichthereby inactivates, reverses or modulates the drug-resistant propertiesin the cancer cells that are otherwise imparted by the increased levelsor overexpression of thioredoxin/glutaredoxin in said cancer cells. Themedicinal and pharmacological inactivation involves the administrationof a Formula (I) compound of the present invention. Any of theaforementioned types of cancer that have increased expression orconcentrations of thioredoxin and/or glutaredoxin are susceptible to andmay benefit from thioredoxin-/glutaredoxin-based intervention by thepresent invention. The present invention also teaches how to optimizethe schedule, dose, and combination of chemotherapy regimens in patientsby the identification in-advance and through-out treatment of thethioredoxin/glutaredoxin levels and the metabolic state within a sampleof cancer cells isolated from the individual patients. Moreover, the useof kits that enable diagnostic and therapeutic optimization of thecompositions and methods of the present invention to further enhance thesurvival outcome and benefit to patients by, for example, thedetermination of the optimum chemotherapeutic drug regimen to utilize.The present invention also teaches how to identify, in advance, thosepatients who would not be likely to benefit from such intervention bythe use of diagnostic kits, thereby allowing other treatment approachesthat may be more clinically efficacious to be pursued. In addition, thediagnostic kits of the present invention allow for continued monitoringof patients and their biochemical responses to treatment.

In brief, the present invention discloses and claims: (i) compositionswhich cause an increase in time of survival in patients with cancer;wherein the cancer either overexpresses thioredoxin or glutaredoxinand/or exhibits or possesses thioredoxin- or glutaredoxin-mediatedresistance to one or more chemotherapeutic agents or interventions; (ii)methods of treatment which cause an increase in the time of survival inpatients with cancer; wherein the cancer either overexpressesthioredoxin or glutaredoxin and/or exhibits or possesses thioredoxin- orglutaredoxin-mediated resistance to one or more chemotherapeutic drugs;(iii) kits for the administration of these compositions to treatpatients with cancer; (iv) methods for quantitatively ascertaining thelevel of expression of thioredoxin or glutaredoxin in patients withcancer; (v) methods of using the level and pattern of expression ofthioredoxin or glutaredoxin in the cancer in the initial diagnosis,planning of subsequent treatment methodologies, and/or ascertaining thepotential treatment responsiveness of the specific cancer of thepatients with cancer; (vi) kits for quantitatively ascertaining thelevel of expression of thioredoxin or glutaredoxin in the cancer ofpatients with cancer; (vii) methods of treatment which cause an increasein time of survival in patients with cancer; wherein the cancer eitheroverexpresses thioredoxin or glutaredoxin and/or exhibits or possessesthioredoxin- or glutaredoxin-mediated resistance to one or morechemotherapeutic drugs and the treatment comprises the administration ofthe chemotherapeutic agents that are sensitive to thioredoxin and/orglutaredoxin overexpression, either of which result in tumor mediateddrug resistance and enhanced angiogenesis; and (viii) methods foroptimizing the schedule, dose, and combination of chemotherapy regimensin patients by ascertaining, in-advance and throughout the treatmentcourse, the thioredoxin levels, glutaredoxin levels and metabolic statein a sample from the patient with cancer.

It should also be noted that, the Japan Phase III non-small cell lungcarcinoma (NSCLC) Clinical Trial and the United States (U.S.) Phase IINSCLC Clinical Trial, that are discussed and described in the presentinvention represent controlled clinical evidence of a survival increasecaused by a thioredoxin and/or glutaredoxin inactivating or modulatingmedicament (that act pharmacologically in the manner of the oxidativemetabolism-affecting Formula (I) compounds of the present invention).These two aforementioned clinical trials will be fully discussed in alater section. However, it is observed from the data from both of thesecontrolled clinical trials that there is a marked increase in patientsurvival, especially in the non-small cell lung carcinoma,adenocarcinoma sub-type patients receiving a Formula (I) compound of thepresent invention. For example, there was an increase in median survivaltime of approximately 138 days (i.e., 4.5 months) and approximately 198days (i.e., 6.5 months) for adenocarcinoma patients in the Tavocept armof the Japan Phase III NSCLC Clinical Trial and the U.S. Phase II NSCLCClinical Trial, respectively.

The compositions of the present invention comprise amedically-sufficient dose of an oxidative metabolism-affecting Formula(I) compound including, but not limited to, the disodium salt of2,2′-dithio-bis-ethane sulfonate, or a pharmaceutically-acceptable saltor analog thereof. The disodium salt of 2,2′-dithio-bis-ethane sulfonatehas also been referred to in the literature as dimesna, Tavocept™, andBNP7787. By way of non-limiting example, disodium 2,2′-dithio-bis-ethanesulfonate (dimesna, Tavocept™, and BNP7787) is a known compound and canbe manufactured by methods known in the art. See, e.g., J. Org. Chem.26:1330-1331 (1961); J. Org. Chem. 59:8239 (1994). In addition, varioussalts and analogs of 2,2′-dithio-bis-ethane sulfonate, as well as otherdithioethers may also be synthesized as outlined in U.S. Pat. No.5,808,160, U.S. Pat. No. 6,160,167 and U.S. Pat. No. 6,504,049, thedisclosures of which are hereby incorporated by reference in theirentirety. Additionally, the compositions of the present invention alsocomprise a medically-sufficient dose of the metabolite of disodium2,2′-dithio-bis-ethane sulfonate, known as 2-mercapto ethane sulfonatesodium (also known in the literature as mesna) and 2-mercapto ethanesulfonate conjugated with a substituent group consisting of:

-Cys, -Homocysteine, -Cys-Gly, -Cys-Glu, -Homocysteine,-Homocysteine-Gly, -Homocysteine-Glu, -Cys-Glu,

wherein R₁ and R₂ are any L- or D-amino acids. These aforementionedheteroconjugate compounds may be synthesized as described in PublishedU.S. Patent Application 2005/0256055, the disclosure of which isincorporated herein, by reference, in its entirety.

The mechanisms of the oxidative metabolism-affecting Formula (I)compounds of the present invention in increasing the survival time ofcancer patients may involve one or more of several novel pharmacologicaland physiological factors, including but not limited to, a prevention,compromise and/or reduction in the normal increase, responsiveness, orin the concentration and/or tumor protective metabolism of variousphysiological cellular thiols; these antioxidants and enzymes areincreased in concentration and/or activity, respectively, in response tothe induction of changes in intracellular oxidative metabolism which maybe caused by exposure to cytotoxic/cytostatic chemotherapeutic agents intumor cells. The Formula (I) compounds of the present invention mayexert an oxidative activity by the intrinsic composition of the moleculeitself (i.e., an oxidized disulfide), as well as by oxidizing freethiols to form oxidized disulfides (i.e., by non-enzymatic SN2-mediatedreactions, wherein attack of a thiol/thiolate upon a disulfide leads tothe scission of the former disulfide which is accompanied by the faciledeparture of a thiol-containing group. As the thiolate group is far morenucleophilic than the corresponding thiol, the attack is believed to bevia the thiolate, however, in some cases the sulfur atom containedwithin an attacking free sulfhydryl group may be the nucleophile), andmay thereby lead to pharmacological depletion and metabolism ofreductive physiological free thiols (e.g., glutathione, cysteine, andhomocysteine).

The Applicant has determined that some of the novel principles governingthese reactions involve the increased (i.e., greater stability of)solvation free energy of the disulfide and free-thiol products that areformed from the reaction; therefore these reactions appear to be largelydriven by the favorable thermodynamics of product formation (i.e.,exothermic reactions). One or more of these pharmacological activitieswill thus have an augmenting (additive or synergistic) effect on thecytotoxic or cytostatic activity of chemotherapeutic agents administeredto patients with cancer, with the additional cytotoxic or cytostaticactivity resulting from the combined administration of the oxidativemetabolism-affecting Formula (I) compounds of the present invention andchemotherapy compounds, thereby leading to: (i) an increase in thecytotoxic and cytoreductive anti-cancer efficacy and decreases intumor-mediated resistance of the various co-administeredchemotherapeutic agents, e.g., platinum- and alkylating agent-based drugefficacy and tumor-mediated drug resistance; (ii) thioredoxininactivation by the Formula (I) compounds of the present invention,thereby increasing apoptotic sensitivity and decreasingmitogenic/cellular replication signaling in cancer cells; (iii) thekilling of cancer cells directly use of a Formula (I) compound,including a key metabolite of disodium 2,2′-dithio-bis-ethane sulfonate(also known in the literature as dimesna, Tavocept™, or BNP7787),2-mercapto ethane sulfonate sodium (also known in the literature asmesna) which possesses intrinsic cytotoxic or cytostatic activity (i.e.,causes apoptosis) in some tumors; and/or (iv) enhancing oxidativemetabolism or compromising the anti-oxidative response of canceroustumor cells, or both, which may thereby enhance their oxidativebiological and physiological state by use of a Formula (I) compound,including 2,2′-dithio-bis-ethane sulfonate compounds (and possibly mesnaor mesna heteroconjugates). This may serve to subsequently increase theamount of oxidative damage in tumor cells exposed to chemotherapyagent(s), thereby enhancing chemotherapy agent-mediated anti-cancercytotoxic, cytostatic, and apoptotic effects. Thus, by enhancingoxidative metabolism and/or reducing or compromising the totalanti-oxidative capacity or responsiveness of cancer tumor cells, anincrease in anti-cancer activity can be achieved—with a resultingincrease in the time of patient survival.

As previously discussed, compositions and formulations comprising theoxidative metabolism-affecting Formula (I) compounds of the presentinvention may be given using any combination of the following threegeneral treatment methods: (i) in a direct inhibitory or inactivatingmanner (i.e., direct chemical interactions that inactivate thioredoxinand/or glutaredoxin) and/or depletive manner (i.e., decreasingthioredoxin and/or glutaredoxin concentrations or production rates) to acancer patient, and thereby increasing the susceptibility of the cancercells to any subsequent administration of any chemotherapeutic agent oragents that may act directly or indirectly through the thioredoxinand/or glutaredoxin-mediated pathways in order to sensitize thepatient's cancer cells and thus to enhance the anti-tumor cytotoxicityof the subsequently-administered chemotherapeutic agent or agents;and/or (ii) in a synergistic manner, where the anti-thioredoxin and/orglutaredoxin therapy is concurrently administered with chemotherapyadministration when a cancer patient begins any chemotherapy cycle, inorder to augment and optimize the pharmacological activity directedagainst thioredoxin and/or glutaredoxin mediated mechanisms presentwhile chemotherapy is being concurrently administered; and/or (iii) in apost-treatment manner (i.e., after the completion of chemotherapy doseadministration or a chemotherapy cycle) in order to maintain thepresence of a pharmacologically-induced depletion, inactivation, ormodulation of thioredoxin and/or glutaredoxin in the patient's cancercells for as long as optimally required. Additionally, theaforementioned compounds may be given in an identical manner to augmentor enhance the anti-cancer activity of a cytotoxic or cytostatic agentby any additionally clinically-beneficial mechanism(s).

The oxidative metabolism-affecting Formula (I) compounds of the presentinvention are compounds which are also capable of increasing thetherapeutic efficacy (i.e., therapeutic index) of a chemotherapeuticdrug, composition, and/or regimen, thus leading to an overall increasein patient survival by, for example: (i) increasing tumor response rate,increasing the time to tumor progression, and delaying/decreasing theonset of metastatic disease; (ii) causing a lack of interference withthe anti-cancer cytotoxic and cytostatic action of an administeredchemotherapeutic agent(s); and (iii) causing a lack of tumordesensitization or drug resistance to the cytotoxic and cytostaticactivity of an administered chemotherapeutic agent(s).

In one embodiment of the present invention, a composition for increasingsurvival time in a patient with cancer is disclosed, wherein the cancer,either: (i) overexpress thioredoxin or glutaredoxin and/or (ii) exhibitevidence of thioredoxin-mediated or glutaredoxin-mediated resistance tothe chemotherapeutic agent or agents used to treat said patient withcancer; is administered in a medically-sufficient dose to the patientwith cancer, either prior to, concomitantly with, or subsequent to theadministration of a chemotherapeutic agent or agents whose cytotoxic orcytostatic activity is adversely by effected by either: (i) theoverexpression of thioredoxin or glutaredoxin and/or (ii)thioredoxin-mediated or glutaredoxin-mediated treatment resistance.

It should be noted that the exhibition of thioredoxin-mediated orglutaredoxin-mediated treatment resistance is described as “evidence of”due to the fact that it is neither expected, nor possible to prove with100% certainty that the cancer cells exhibit thioredoxin-mediated orglutaredoxin-mediated treatment resistance, prior to the treatment ofthe patient. By way of non-limiting example, the current use of, e.g.,florescence in situ hybridization (FISH) or immunohistochemistry (IHC)to guide treatment decisions for HER2/neu-based therapy are predicatedupon the probability of the overexpression/increased concentrations ofHER2/neu being correlated with the probability of a therapeuticresponse. Such expectation of a therapeutic response is not 100%certain, and is related to many factors, not the least of which is thediagnostic accuracy of the test utilized which, in turn, is also limitedby the sampling of the tumor and various other factors (e.g., laboratorymethodology/technique, reagent quality, and the like).

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of any cancerwhich either: (i) overexpresses thioredoxin or glutaredoxin and/or (ii)exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents being used to treatsaid patient with cancer.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of: lung cancer,colorectal cancer, gastric cancer, esophageal cancer, ovarian cancer,cancer of the biliary tract, gallbladder cancer, cervical cancer, breastcancer, endometrial cancer, vaginal cancer, prostate cancer, uterinecancer, hepatic cancer, pancreatic cancer, and adenocarcinoma.

In one embodiment of the present invention, a composition for increasingsurvival time in a patient with non-small cell lung carcinoma isdisclosed, wherein the non-small cell lung carcinoma, either: (i)overexpresses thioredoxin or glutaredoxin and/or (ii) exhibits evidenceof thioredoxin-mediated or glutaredoxin-mediated resistance to thechemotherapeutic agent or agents used to treat said patient withnon-small cell lung carcinoma; is administered in a medically-sufficientdose to the patient with non-small cell lung carcinoma, either prior to,concomitantly with, or subsequent to the administration of achemotherapeutic agent or agents whose cytotoxic or cytostatic activityis adversely by effected by either: (i) the overexpression ofthioredoxin or glutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance.

In another embodiment of the present invention, a composition forincreasing survival time in a patient with adenocarcinoma is disclosed,wherein the adenocarcinoma, either: (i) overexpresses thioredoxin orglutaredoxin and/or (ii) exhibits evidence of thioredoxin-mediated orglutaredoxin-mediated resistance to the chemotherapeutic agent or agentsused to treat said patient with adenocarcinoma; is administered in amedically-sufficient dose to the patient with adenocarcinoma, eitherprior to, concomitantly with, or subsequent to the administration of achemotherapeutic agent or agents whose cytotoxic or cytostatic activityis adversely by effected by either: (i) the overexpression ofthioredoxin or glutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance.

In one embodiment of the present invention, a method of increasingsurvival time in a patient with cancer is disclosed, wherein the cancer,either: (i) overexpresses thioredoxin or glutaredoxin and/or (ii)exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents used to treat saidpatient with non-small cell lung carcinoma; wherein said methodcomprises the administration of a medically-sufficient dose of a Formula(I) compound to said patient with cancer either prior to, concomitantlywith, or subsequent to the administration of a chemotherapeutic agent oragents whose cytotoxic or cytostatic activity is adversely by effectedby either: (i) the overexpression of thioredoxin or glutaredoxin and/or(ii) the thioredoxin-mediated or glutaredoxin-mediated treatmentresistance.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of any cancerwhich either: (i) overexpresses thioredoxin or glutaredoxin and/or (ii)exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents being used to treatsaid patient with cancer.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of: lung cancer,colorectal cancer, gastric cancer, esophageal cancer, ovarian cancer,cancer of the biliary tract, gallbladder cancer, cervical cancer, breastcancer, endometrial cancer, vaginal cancer, prostate cancer, uterinecancer, hepatic cancer, pancreatic cancer, and adenocarcinoma.

In another embodiment of the present invention, a method of increasingsurvival time in a patient with non-small cell lung carcinoma isdisclosed, wherein the non-small lung carcinoma, either: (i)overexpresses thioredoxin or glutaredoxin and/or (ii) exhibits evidenceof thioredoxin-mediated or glutaredoxin-mediated resistance to thechemotherapeutic agent or agents used to treat said patient withnon-small cell lung carcinoma; wherein said method comprises theadministration of a medically-sufficient dose of a Formula (I) compoundto said patient with non-small cell lung carcinoma either prior to,concomitantly with, or subsequent to the administration of achemotherapeutic agent or agents whose cytotoxic or cytostatic activityis adversely affected by either: (i) the overexpression of thioredoxinor glutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance.

In yet another embodiment of the present invention, a method ofincreasing survival time in a patient with adenocarcinoma is disclosed,wherein the adenocarcinoma, either: (i) overexpresses thioredoxin orglutaredoxin and/or (ii) exhibits evidence of thioredoxin-mediated orglutaredoxin-mediated resistance to the chemotherapeutic agent or agentsused to treat said patient with adenocarcinoma; wherein said methodcomprises the administration of a medically-sufficient dose of anoxidative metabolism-affecting Formula (I) compound to said patient withadenocarcinoma either prior to, concomitantly with, or subsequent to theadministration of a chemotherapeutic agent or agents whose cytotoxic orcytostatic activity is adversely affected by either: (i) theoverexpression of thioredoxin or glutaredoxin and/or (ii) thethioredoxin-mediated or glutaredoxin-mediated treatment resistance.

In one embodiment of the present invention, a kit comprising anoxidative metabolism-affecting Formula (I) compound for administration,and instructions for administering said Formula (I) compound to apatient with cancer in an amount sufficient to cause an increase in thesurvival time of said patient with cancer who is receiving achemotherapeutic agent or agents whose cytotoxic or cytostatic activityis adversely affected by either: (i) the overexpression of thioredoxinor glutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance, is disclosed.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of any cancerwhich either: (i) overexpresses thioredoxin or glutaredoxin and/or (ii)exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents being used to treatsaid patient with cancer.

In another embodiment of the present invention, a kit comprising anoxidative metabolism-affecting Formula (I) compound for administration,and instructions for administering said Formula (I) compound to apatient with non-small cell lung carcinoma in an amount sufficient tocause an increase in the survival time of said patient who is receivinga chemotherapeutic agent or agents whose cytotoxic or cytostaticactivity is adversely affected by either: (i) the overexpression ofthioredoxin or glutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance, is disclosed.

In yet another embodiment, a kit comprising a Formula (I) compound foradministration, and instructions for administering said Formula (I)compound to a patient with adenocarcinoma in an amount sufficient tocause an increase in the survival time of said patient who is receivinga chemotherapeutic agent or agents whose cytotoxic or cytostaticactivity is adversely affected by either: (i) the overexpression ofthioredoxin or glutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance, is disclosed.

In one embodiment of the present invention, a method for quantitativelyascertaining the level of thioredoxin or glutaredoxin DNA, mRNA, orprotein in cells which have been isolated from a patient who issuspected of having cancer or has already been diagnosed with cancer isdisclosed; wherein the method used to identify levels of thioredoxin orglutaredoxin is selected from the group consisting of: fluorescence insitu hybridization (FISH), nucleic acid microarray analysis,immunohistochemistry (IHC), and radioimmunoassay (RIA).

In another embodiment, the method is used in the initial diagnosis, theplanning of subsequent treatment methodologies, and/or determining thepotential aggressiveness of cancer growth in a patient suffering from atype of cancer in which the cells comprising the cancer either: (i)overexpress thioredoxin or glutaredoxin and/or (ii) exhibit evidence ofthioredoxin-mediated or glutaredoxin-mediated treatment resistance tothe chemotherapeutic agents or agents already being administered to thepatient with cancer.

In still another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of: lung cancer,colorectal cancer, gastric cancer, esophageal cancer, ovarian cancer,cancer of the biliary tract, gallbladder cancer, cervical cancer, breastcancer, endometrial cancer, vaginal cancer, prostate cancer, uterinecancer, hepatic cancer, pancreatic cancer, and adenocarcinoma.

In one embodiment of the present invention, a kit with instructions forquantitatively ascertaining the level of thioredoxin or glutaredoxinDNA, mRNA, or protein in cells which have been isolated from a patientwho is suspected of having cancer or has already been diagnosed withcancer is disclosed; wherein the kit uses a method to identify levels ofthioredoxin or glutaredoxin which is selected from the group consistingof: fluorescence in situ hybridization (FISH), nucleic acid microarrayanalysis, immunohistochemistry (IHC), and radioimmunoassay (RIA).

In yet another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of: lung cancer,colorectal cancer, gastric cancer, esophageal cancer, ovarian cancer,cancer of the biliary tract, gallbladder cancer, cervical cancer, breastcancer, endometrial cancer, vaginal cancer, prostate cancer, uterinecancer, hepatic cancer, pancreatic cancer, and adenocarcinoma.

In another embodiment of the present invention, a method for increasingsurvival time in a patient with cancer is disclosed, wherein saidcancer, either: (i) overexpresses thioredoxin or glutaredoxin and/or(ii) exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents used to treat saidpatient with cancer; wherein said method comprises the administration ofa medically-sufficient dose of a Formula (I) compound to said patientwith cancer either prior to, concomitantly with, or subsequent to theadministration of the chemotherapeutic agents cisplatin and docetaxel;wherein the cytotoxic or cytostatic activity of the chemotherapeuticagents is adversely affected by either: (i) the overexpression ofthioredoxin or glutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from any cancer that either: (i)overexpresses thioredoxin or glutaredoxin and/or (ii) exhibits evidenceof thioredoxin-mediated or glutaredoxin-mediated treatment resistance tothe chemotherapeutic agents or agents already being administered to saidpatient with cancer.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of lung cancer,colorectal cancer, gastric cancer, esophageal cancer, ovarian cancer,cancer of the biliary tract, gallbladder cancer, cervical cancer, breastcancer, endometrial cancer, vaginal cancer, prostate cancer, uterinecancer, hepatic cancer, pancreatic cancer, and adenocarcinoma.

In one embodiment of the present invention, a method for increasingsurvival time in a cancer patient with non-small cell lung carcinoma isdisclosed, wherein the non-small cell lung carcinoma, either: (i)overexpresses thioredoxin or glutaredoxin and/or (ii) exhibits evidenceof thioredoxin-mediated or glutaredoxin-mediated resistance to thechemotherapeutic agent or agents used to treat said patient withnon-small cell lung carcinoma; wherein said method comprises theadministration of a medically-sufficient dose of a Formula (I) compoundto said patient with non-small cell lung carcinoma either prior to,concomitantly with, or subsequent to the administration of thechemotherapeutic agents cisplatin and docetaxel; wherein the cytotoxicor cytostatic activity of said chemotherapeutic agents is adverselyaffected by either: (i) the overexpression of thioredoxin orglutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance.

In another embodiment, a method for increasing survival time in a cancerpatient with adenocarcinoma is disclosed, wherein the adenocarcinoma,either: (i) overexpresses thioredoxin or glutaredoxin and/or (ii)exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents used to treat saidpatient with adenocarcinoma; wherein said method comprises theadministration of a medically-sufficient dose of a Formula (I) compoundto said patient with adenocarcinoma either prior to, concomitantly with,or subsequent to the administration of the chemotherapeutic agentscisplatin and docetaxel; wherein the cytotoxic or cytostatic activity ofsaid chemotherapeutic agents is adversely affected by either: (i) theoverexpression of thioredoxin or glutaredoxin and/or (ii) thethioredoxin-mediated or glutaredoxin-mediated treatment resistance.

In yet another embodiment, the method is comprised of (i) theadministration of docetaxel at a dose of 75 mg/m² which is givenintravenously over a period of approximately 1 hour; (ii) theadministration of docetaxel in step (i) is immediately followed by theadministration of disodium 2,2′-dithio-bis-ethane sulfonate (Tavocept™)at a dose of approximately 40 grams which is given intravenously over aperiod of approximately 30 minutes; and (iii) the administration ofdisodium 2,2′-dithio-bis-ethane sulfonate (Tavocept™) in step (ii) isimmediately followed by the administration of cisplatin at a dose of 75mg/m² which is given intravenously over a period of approximately 1 hourwith concomitant sufficient intravenous hydration; wherein steps(i)-(iii) constitute a single chemotherapy cycle which can be repeatedevery two weeks, for up to a total of six cycles.

In another embodiment, a kit comprising a Formula (I) compound foradministration, and instructions for administering said Formula (I)compound to a patient with any medical condition or disease whereinthere is overexpression of thioredoxin or glutaredoxin is disclosed,wherein said kit comprises the administration of a medically-sufficientdose of a Formula (I) compound, and wherein the overexpression ofthioredoxin or glutaredoxin causes deleterious physiological effects insaid patient.

In various embodiments of the present, the composition is a Formula (I)compound having the structural formula:

X—S—S—R₁-R₂:

wherein;

-   -   R₁ is a lower alkylene, wherein R₁ is optionally substituted by        a member of the group consisting of: lower alkyl, aryl, hydroxy,        alkoxy, aryloxy, mercapto, alkylthio or arylthio, for a        corresponding hydrogen atom, or

-   -   R₂ and R₄ is sulfonate or phosphonate;    -   R₅ is hydrogen, hydroxy, or sulfhydryl;    -   m is 0, 1, 2, 3, 4, 5, or 6; and    -   X is a sulfur-containing amino acid or a peptide consisting of        from 2-10 amino acids;    -   or wherein X is a member of the group consisting of lower        thioalkyl (lower mercapto alkyl), lower alkylsulfonate, lower        alkylphosphonate, lower alkenylsulfonate, lower alkyl, lower        alkenyl, lower alkynyl, aryl, alkoxy, aryloxy, mercapto,        alkylthio or hydroxy for a corresponding hydrogen atom; and        pharmaceutically-acceptable salts, prodrugs, analogs,        conjugates, hydrates, solvates, polymorphs, stereoisomers        (including diastereoisomers and enantiomers) and tautomers        thereof.

In other embodiments of the present invention, the composition is apharmaceutically-acceptable disodium salt of a Formula (I) compound. Instill other embodiments, the composition of the present invention is/area pharmaceutically-acceptable salt(s) of a Formula (I) compound whichinclude, for example: (i) a monosodium salt; (ii) a sodium potassiumsalt; (iii) a dipotassium salt; (iv) a calcium salt; (v) a magnesiumsalt; (vi) a manganese salt; (vii) a monopotassium salt; and (viii) anammonium salt. It should be noted that mono- and di-potassium salts of2,2′-dithio-bis-ethane sulfonate and/or an analog thereof areadministered to a subject if the total dose of potassium administered atany given point in time is not greater than 100 Meq. and the subject isnot hyperkalemic and does not have a condition that would predispose thesubject to hyperkalemia (e.g., renal failure).

In embodiments of the present invention, the composition is disodium2,2′-dithio-bis-ethane sulfonate (also known in the literature asTavocept™, BNP7787, and dimesna).

In yet other embodiments, the composition is 2-mercapto-ethane sulfonateor 2-mercapto-ethane sulfonate conjugated as a disulfide with asubstituent group selected from the group consisting of:

-Cys, -Homocysteine, -Cys-Gly, -Cys-Glu, -Homocysteine,-Homocysteine-Gly, -Homocysteine-Glu, -Cys-Glu,

wherein R₁ and R₂ are any L- or D-amino acids.

In other embodiments, the chemotherapy agent or agents administered areselected from the group consisting of fluoropyrimidines; pyrimidinenucleosides; purine nucleosides; anti-folates, platinum agents;anthracyclines/anthracenediones; epipodophyllotoxins; camptothecins;hormones; hormonal complexes; antihormonals; enzymes, proteins, peptidesand polyclonal and/or monoclonal antibodies; vinca alkaloids; taxanes;epothilones; antimicrotubule agents; alkylating agents; antimetabolites;topoisomerase inhibitors; aziridine-containing compounds; antivirals;and various other cytotoxic and cytostatic agents.

In embodiments of the present invention, the chemotherapy agent oragents are selected from the group consisting of: cisplatin,carboplatin, oxaliplatin, satraplatin, picoplatin, tetraplatin,platinum-DACH, and analogs and derivatives thereof.

In other embodiments, the chemotherapy agent or agents are selected fromthe group consisting of: docetaxel, paclitaxel, polyglutamylated formsof paclitaxel, liposomal paclitaxel, and analogs and derivativesthereof.

In yet other embodiments of the present invention, the chemotherapyagents are docetaxel and cisplatin.

Furthermore, in brief, the present invention discloses and claims: (i)compositions, methods, and kits which lead to an increase in patientsurvival time in cancer patients receiving chemotherapy; (ii)compositions and methods which cause cytotoxic or apoptotic potentiationof the anti-cancer activity of chemotherapeutic agents; (iii)compositions and methods for maintaining or stimulating hematologicalfunction in patients in need thereof, including those patients sufferingfrom cancer; (iv) compositions and methods for maintaining orstimulating erythropoietin function or synthesis in patients in needthereof, including those patients suffering from cancer; (v)compositions and methods for mitigating or preventing anemia in patientsin need thereof, including those patients suffering from cancer; (vi)compositions and methods for maintaining or stimulating pluripotent,multipotent, and unipotent normal stem cell function or synthesis inpatients in need thereof, including those patients suffering fromcancer; (vii) compositions and methods which promote the arrest orretardation of tumor progression in cancer patients receivingchemotherapy; (viii) compositions and methods for increasing patientsurvival and/or delaying tumor progression while maintaining orimproving the quality of life in a cancer patient receivingchemotherapy; (ix) novel methods of the administration of taxane andplatinum medicaments and a Formula (I) compound of the present inventionto a cancer patient; and (x) kits to achieve one or more of theaforementioned physiological effects in a patient in need thereof,including those patients suffering from cancer.

In one embodiment, a patient suffering from lung cancer treated withtaxane and/or platinum medicaments is given a medically sufficientdosage of a Formula (I) compound so as to increase patient survival timein said patient suffering from lung cancer.

In another embodiment, the lung cancer is non-small cell lung carcinoma.

In another embodiment, the increase in patient survival time in saidpatient suffering from lung cancer and treated with a Formula (I)compound is expected to be at least 30 days longer than the expectedsurvival time if said patient was not treated with a Formula (I)compound.

In yet another embodiment, a patient suffering from lung cancer wastreated with paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks, wherein the dose of paclitaxel ranged fromapproximately 160 mg/m² to approximately 190 mg/m², the dose of aFormula (I) compound ranged from approximately 14 g/m² to approximately22 g/m², and the dose of cisplatin ranged from approximately 60 mg/m² toapproximately 100 mg/m², wherein said administration of paclitaxel, aFormula (I) compound, and cisplatin once every 2-4 weeks was repeated atleast once.

In still another embodiment, a patient suffering from lung cancer wastreated with paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks, wherein the dose of paclitaxel was approximately 175mg/m², the dose of a Formula (I) compound was approximately 18.4 g/m²,and the dose of cisplatin ranged from approximately 75 mg/m² toapproximately 85 mg/m², wherein said administration of paclitaxel, aFormula (I) compound, and cisplatin once every 3 weeks was repeated for6 cycles.

In another embodiment, the patients suffering from lung cancer were maleor female and smokers or non-smokers.

In one embodiment, a patient suffering from adenocarcinoma treated withtaxane and/or platinum medicaments is given a medically sufficientdosage of a Formula (I) compound so as to increase patient survival timein said patient suffering from adenocarcinoma.

In another embodiment, the increase in patient survival time in saidpatient suffering from adenocarcinoma and treated with a Formula (I)compound is expected to be at least 30 days longer than the expectedsurvival time if said patient was not treated with a Formula (I)compound.

In yet another embodiment, a patient suffering from adenocarcinoma istreated with paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks, wherein the dose of paclitaxel ranged fromapproximately 160 mg/m² to approximately 190 mg/m², the dose of aFormula (I) compound ranged from approximately 14 g/m² to approximately22 g/m², and the dose of cisplatin ranged from approximately 60 mg/m² toapproximately 100 mg/m², wherein said administration of paclitaxel, aFormula (I) compound, and cisplatin once every 2-4 weeks was repeated atleast once.

In still another embodiment, a patient suffering from adenocarcinoma istreated with paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks, wherein the dose of paclitaxel was approximately 175mg/m², the dose of a Formula (I) compound was approximately 18.4 g/m²,and the dose of cisplatin ranged from approximately 75 mg/m² toapproximately 85 mg/m², wherein said administration of paclitaxel, aFormula (I) compound, and cisplatin once every 3 weeks was repeated for6 cycles.

In another embodiment, the patients suffering from adenocarcinoma weremale or female and smokers or non-smokers.

In one embodiment, a patient suffering from lung cancer treated withtaxane and platinum medicaments is given a medically sufficient dosageof a Formula (I) compound so as to potentiate the chemotherapeuticeffect in said patient suffering from lung cancer.

In another embodiment, the lung cancer is non-small cell lung carcinoma.

In yet another embodiment, the chemotherapeutic effect is potentiated ina patient suffering from lung cancer treated with paclitaxel, a Formula(I) compound, and cisplatin once every 2-4 weeks, wherein the dose ofpaclitaxel ranged from approximately 160 mg/m² to approximately 190mg/m², the dose of a Formula (I) compound ranged from approximately 14g/m² to approximately 22 g/m², and the dose of cisplatin ranged fromapproximately 60 mg/m² to approximately 100 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks was repeated at least once.

In still another embodiment, the chemotherapeutic effect is potentiatedin a patient suffering from lung cancer treated with paclitaxel, aFormula (I) compound, and cisplatin once every 3 weeks, wherein the doseof paclitaxel was approximately 175 mg/m², the dose of a Formula (I)compound was approximately 18.4 g/m², and the dose of cisplatin rangedfrom approximately 75 mg/m² to approximately 85 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from lung cancer were maleor female and smokers or non-smokers.

In one embodiment, the chemotherapeutic effect is potentiated in apatient suffering from adenocarcinoma who is treated with taxane andplatinum medicaments and is also given a medically sufficient dosage ofa Formula (I) compound so as to increase patient survival time in saidpatient suffering from adenocarcinoma.

In yet another embodiment, the chemotherapeutic effect is potentiated ina patient suffering from adenocarcinoma treated with paclitaxel, aFormula (I) compound, and cisplatin once every 2-4 weeks, wherein thedose of paclitaxel ranged from approximately 160 mg/m² to approximately190 mg/m², the dose of a Formula (I) compound ranged from approximately14 g/m² to approximately 22 g/m², and the dose of cisplatin ranged fromapproximately 60 mg/m² to approximately 100 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks was repeated at least once.

In still another embodiment, the chemotherapeutic effect is potentiatedin a patient suffering from adenocarcinoma treated with paclitaxel, aFormula (I) compound, and cisplatin once every 3 weeks, wherein the doseof paclitaxel was approximately 175 mg/m², the dose of a Formula (I)compound was approximately 18.4 g/m², and the dose of cisplatin rangedfrom approximately 75 mg/m² to approximately 85 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from adenocarcinoma weremale or female and smokers or non-smokers.

In one embodiment, hematological function is maintained or stimulated ina patient in need thereof, by providing to said patient a compositioncomprised of a Formula (I) compound in a medically sufficient dosage.

In one embodiment, a patient suffering from lung cancer treated withtaxane and/or platinum medicaments is given a medically sufficientdosage of a Formula (I) compound so as to maintain or stimulatehematological function in said patient suffering from lung cancer.

In another embodiment, the lung cancer is non-small cell lung carcinoma.

In yet another embodiment, the hematological function is maintained orstimulated in a patient suffering from lung cancer treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 2-4 weeks,wherein the dose of paclitaxel ranged from approximately 160 mg/m² toapproximately 190 mg/m², the dose of a Formula (I) compound ranged fromapproximately 14 g/m² to approximately 22 g/m², and the dose ofcisplatin ranged from approximately 60 mg/m² to approximately 100 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 2-4 weeks was repeated at least once.

In still another embodiment, the hematological function is maintained orstimulated in a patient suffering from lung cancer treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 3 weeks,wherein the dose of paclitaxel was approximately 175 mg/m², the dose ofa Formula (I) compound was approximately 18.4 g/m², and the dose ofcisplatin ranged from approximately 75 mg/m² to approximately 85 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from lung cancer were maleor female and smokers or non-smokers.

In one embodiment, the hematological function is maintained orstimulated in a patient suffering from adenocarcinoma who is treatedwith taxane and/or platinum medicaments and is also given a medicallysufficient dosage of a Formula (I) compound so as to maintain orstimulate hematological function in said patient suffering fromadenocarcinoma.

In yet another embodiment, the hematological function is maintained orstimulated in a patient suffering from adenocarcinoma treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 2-4 weeks,wherein the dose of paclitaxel ranged from approximately 160 mg/m² toapproximately 190 mg/m², the dose of a Formula (I) compound ranged fromapproximately 14 g/m² to approximately 22 g/m², and the dose ofcisplatin ranged from approximately 60 mg/m² to approximately 100 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 2-4 weeks was repeated at least once.

In still another embodiment, the hematological function is maintained orstimulated in a patient suffering from adenocarcinoma treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 3 weeks,wherein the dose of paclitaxel was approximately 175 mg/m², the dose ofa Formula (I) compound was approximately 18.4 g/m², and the dose ofcisplatin ranged from approximately 75 mg/m² to approximately 85 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from adenocarcinoma weremale or female and smokers or non-smokers.

In one embodiment, erythropoietin function or synthesis or homeostaticfunction of erythropoiesis is maintained or stimulated in a patient inneed thereof, by providing to said patient a composition comprised of aFormula (I) compound in a medically sufficient dosage.

In one embodiment, a patient suffering from lung cancer treated withtaxane and/or platinum medicaments is given a medically sufficientdosage of a Formula (I) compound so as to maintain or stimulateerythropoietin function or synthesis or homeostatic function oferythropoiesis in said patient suffering from lung cancer.

In another embodiment, the lung cancer is non-small cell lung carcinoma.

In yet another embodiment, the erythropoietin function or synthesis orhomeostatic function of erythropoiesis is maintained or stimulated in apatient suffering from lung cancer treated with paclitaxel, a Formula(I) compound, and cisplatin once every 2-4 weeks, wherein the dose ofpaclitaxel ranged from approximately 160 mg/m² to approximately 190mg/m², the dose of a Formula (I) compound ranged from approximately 14g/m² to approximately 22 g/m², and the dose of cisplatin ranged fromapproximately 60 mg/m² to approximately 100 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks was repeated at least once.

In still another embodiment, the erythropoietin function or synthesis orhomeostatic function of erythropoiesis is maintained or stimulated in apatient suffering from lung cancer treated with paclitaxel, a Formula(I) compound, and cisplatin once every 3 weeks, wherein the dose ofpaclitaxel was approximately 175 mg/m², the dose of a Formula (I)compound was approximately 18.4 g/m², and the dose of cisplatin rangedfrom approximately 75 mg/m² to approximately 85 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from lung cancer were maleor female and smokers or non-smokers.

In one embodiment, the erythropoietin function or synthesis orhomeostatic function of erythropoiesis is maintained or stimulated in apatient suffering from adenocarcinoma who is treated with taxane and/orplatinum medicaments and is also given a medically sufficient dosage ofa Formula (I) compound so as to maintain or stimulate erythropoietinfunction or synthesis or homeostatic function of erythropoiesis in saidpatient suffering from adenocarcinoma.

In yet another embodiment, the erythropoietin function or synthesis orhomeostatic function of erythropoiesis is maintained or stimulated in apatient Suffering from adenocarcinoma treated with paclitaxel, a Formula(I) compound, and cisplatin once every 2-4 weeks, wherein the dose ofpaclitaxel ranged from approximately 160 mg/m² to approximately 190mg/m², the dose of a Formula (I) compound ranged from approximately 14g/m² to approximately 22 g/m², and the dose of cisplatin ranged fromapproximately 60 mg/m² to approximately 100 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks was repeated at least once.

In still another embodiment, the erythropoietin function or synthesis orhomeostatic function of erythropoiesis is maintained or stimulated in apatient suffering from adenocarcinoma treated with paclitaxel, a Formula(I) compound, and cisplatin once every 3 weeks, wherein the dose ofpaclitaxel was approximately 175 mg/m², the dose of a Formula (I)compound was approximately 18.4 g/m², and the dose of cisplatin rangedfrom approximately 75 mg/m² to approximately 85 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from adenocarcinoma weremale or female and smokers or non-smokers.

In one embodiment, anemia is mitigated or prevented in a patient in needthereof, by providing to said patient a composition comprised of aFormula (I) compound in a medically sufficient dosage.

In one embodiment, a patient suffering from lung cancer treated withtaxane and/or platinum medicaments is given a medically sufficientdosage of a Formula (I) compound so as to mitigate or preventchemotherapy-induced anemia in said patient suffering from lung cancer.

In another embodiment, the lung cancer is non-small cell lung carcinoma.

In yet another embodiment, chemotherapy-induced anemia is mitigated orprevented in a patient suffering from lung cancer treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 2-4 weeks,wherein the dose of paclitaxel ranged from approximately 160 mg/m² toapproximately 190 mg/m², the dose of a Formula (I) compound ranged fromapproximately 14 g/m² to approximately 22 g/m², and the dose ofcisplatin ranged from approximately 60 mg/m² to approximately 100 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 2-4 weeks was repeated at least once.

In still another embodiment, chemotherapy-induced anemia is mitigated orprevented in a patient suffering from lung cancer treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 3 weeks,wherein the dose of paclitaxel was approximately 175 mg/m², the dose ofa Formula (I) compound was approximately 18.4 g/m², and the dose ofcisplatin ranged from approximately 75 mg/m² to approximately 85 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from lung cancer were maleor female and smokers or non-smokers.

In one embodiment, chemotherapy-induced anemia is mitigated or preventedin a patient suffering from adenocarcinoma who is treated with taxaneand/or platinum medicaments and is also given a medically sufficientdosage of a Formula (I) compound so as to mitigate or preventchemotherapy-induced anemia.

In yet another embodiment, chemotherapy-induced anemia is mitigated orprevented in a patient suffering from adenocarcinoma treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 2-4 weeks,wherein the dose of paclitaxel ranged from approximately 160 mg/m² toapproximately 190 mg/m², the dose of a Formula (I) compound ranged fromapproximately 14 g/m² to approximately 22 g/m², and the dose ofcisplatin ranged from approximately 60 mg/m² to approximately 100 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 2-4 weeks was repeated at least once.

In still another embodiment, chemotherapy-induced anemia is mitigated orprevented in a patient suffering from adenocarcinoma treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 3 weeks,wherein the dose of paclitaxel was approximately 175 mg/m², the dose ofa Formula (I) compound was approximately 18.4 g/m², and the dose ofcisplatin ranged from approximately 75 mg/m² to approximately 85 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from adenocarcinoma weremale or female and smokers or non-smokers.

In one embodiment, pluripotent, multipotent, and unipotent normal stemcell function or synthesis is maintained or stimulated in a patient inneed thereof, by providing to said patient a composition comprised of aFormula (I) compound in a medically sufficient dosage.

In one embodiment, a patient suffering from lung cancer treated withtaxane and/or platinum medicaments is given a medically sufficientdosage of a Formula (I) compound so as to maintain or stimulatepluripotent, multipotent, and unipotent normal stem cell function orsynthesis in said patient suffering from lung cancer.

In another embodiment, the lung cancer is non-small cell lung carcinoma.

In yet another embodiment, pluripotent, multipotent, and unipotentnormal stem cell function or synthesis is maintained or stimulated in apatient suffering from lung cancer treated with paclitaxel, a Formula(I) compound, and cisplatin once every 2-4 weeks, wherein the dose ofpaclitaxel ranged from approximately 160 mg/m² to approximately 190mg/m², the dose of a Formula (I) compound ranged from approximately 14g/m² to approximately 22 g/m², and the dose of cisplatin ranged fromapproximately 60 mg/m² to approximately 100 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks was repeated at least once.

In still another embodiment, pluripotent, multipotent, and unipotentnormal stem cell function or synthesis is maintained or stimulated in apatient suffering from lung cancer treated with paclitaxel, a Formula(I) compound, and cisplatin once every 3 weeks, wherein the dose ofpaclitaxel was approximately 175 mg/m², the dose of a Formula (I)compound was approximately 18.4 g/m², and the dose of cisplatin rangedfrom approximately 75 mg/m² to approximately 85 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from lung cancer were maleor female and smokers or non-smokers.

In one embodiment, pluripotent, multipotent, and unipotent normal stemcell function or synthesis is maintained or stimulated in a patientsuffering from adenocarcinoma who is treated with taxane and/or platinummedicaments and is also given a medically sufficient dosage of a Formula(I) compound so as to maintain or stimulate pluripotent, multipotent,and unipotent normal stem cell function or synthesis in said patientsuffering from adenocarcinoma.

In yet another embodiment, pluripotent, multipotent, and unipotentnormal stem cell function or synthesis is maintained or stimulated in apatient suffering from adenocarcinoma treated with paclitaxel, a Formula(I) compound, and cisplatin once every 2-4 weeks, wherein the dose ofpaclitaxel ranged from approximately 160 mg/m² to approximately 190mg/m², the dose of a Formula (I) compound ranged from approximately 14g/m² to approximately 22 g/m², and the dose of cisplatin ranged fromapproximately 60 mg/m² to approximately 100 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks was repeated at least once.

In still another embodiment, pluripotent, multipotent, and unipotentnormal stem cell function or synthesis is maintained or stimulated in apatient suffering from adenocarcinoma treated with paclitaxel, a Formula(I) compound, and cisplatin once every 3 weeks, wherein the dose ofpaclitaxel was approximately 175 mg/m², the dose of a Formula (I)compound was approximately 18.4 g/m², and the dose of cisplatin rangedfrom approximately 75 mg/m² to approximately 85 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from adenocarcinoma weremale or female and smokers or non-smokers.

In another embodiment, the Formula (I) compounds increase patientsurvival and/or delay tumor progression while maintaining or improvingthe quality of life of said patients diagnosed with lung cancer who arebeing treated with the taxane and/or platinum medicaments of the presentinvention.

In another embodiment, the lung cancer is non-small cell lung carcinoma.

In another embodiment, the Formula (I) compounds increase patientsurvival and/or delay tumor progression while maintaining or improvingthe quality of life of said patients diagnosed with adenocarcinoma whoare being treated with the taxane and/or platinum medicaments of thepresent invention.

In another embodiment, the patients suffering from adenocarcinoma weremale or female and smokers or non-smokers.

In another embodiment, the platinum medicaments of the present inventioninclude cisplatin, oxaliplatin, carboplatin, satraplatin, andderivatives and analogs thereof.

In another embodiment, the taxane medicament is selected from the groupconsisting of docetaxel, paclitaxel, paclitaxel derivatives,polyglutamylated forms of paclitaxel, liposomal paclitaxel, andderivatives and analogs thereof.

In still another embodiment, the compositions of Formula (I) include2,2′-dithio-bis-ethane sulfonate, a pharmaceutically-acceptable saltthereof, and/or an analog thereof, as well as prodrugs, analogs,conjugates, hydrates, solvates and polymorphs, as well as stereoisomers(including diastereoisomers and enantiomers) and tautomers of suchcompounds.

In still another embodiment, the dose rate of the taxane and platinummedicaments ranged from approximately 10-20 mg/m²/day and the dose rateof a Formula (I) compound ranged from approximately 4.1-41.0 g/m² perday; the concentration of the taxane and platinum medicaments and/orFormula (I) compounds is at least 0.01 mg/mL; the infusion time of thetaxane and platinum medicaments and/or Formula (I) compounds is fromapproximately 5 minutes to approximately 24 hours, and can be repeatedas needed and tolerated in a given patient; the schedule ofadministration of the taxane and platinum medicaments and/or Formula (I)compounds is every 2-8 weeks.

In another embodiment, a kit comprising a Formula (I) compound foradministration to a patient, and instructions for administering saidFormula (I) compound in an amount sufficient to cause one or more of thephysiological effects selected from the group consisting of: increasingpatient survival time of said cancer patient receiving taxane and/orplatinum medicaments; causing a cytotoxic or apoptotic potentiation ofthe chemotherapeutic effects of said taxane and platinum medicaments;maintaining or stimulating hematological function in said patient,including said patient with cancer receiving chemotherapy; maintainingor stimulating erythropoietin function or synthesis in said patient,including said patient with cancer receiving chemotherapy; mitigating orpreventing anemia in said patient, including said patient with cancerreceiving chemotherapy; maintaining or stimulating pluripotent,multipotent, and unipotent normal stem cell function or synthesis insaid patient, including said patient with cancer receiving chemotherapy;promoting the arrest or retardation of tumor progression in said cancerpatient receiving taxane and platinum medicaments; and/or increasingpatient survival and/or delaying tumor progression while maintaining orimproving the quality of life in said cancer patient receiving taxaneand platinum medicaments.

In another embodiment, the cancer patient has lung cancer.

In yet another embodiment, the lung cancer is non-small cell lungcancer.

In still another embodiment, the cancer patient has an adenocarcinoma.

In one embodiment, the kit further contains instructions foradministering a taxane medicament and a platinum medicament selectedfrom the group consisting of cisplatin, oxaliplatin, carboplatin,satraplatin, and derivatives and analogs thereof.

In another embodiment, the kit further contains instructions foradministering a platinum medicament and a taxane medicament selectedfrom the group consisting of docetaxel, paclitaxel, polyglutamylatedforms of paclitaxel, liposomal paclitaxel, and derivatives and analogsthereof.

In yet another embodiment, the platinum and taxane medicaments arecisplatin and paclitaxel.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the involvement of (reduced) glutaredoxin inpromoting cell growth and/or stimulating cell proliferation via severalmetabolic pathways. The glutaredoxin system consists of glutaredoxin,glutathione and glutathione reductase. It should be noted, however, thatglutaredoxin is also involved in many other intracellular pathways.

FIG. 2 illustrates the coupled glutaredoxin (Gxr)/glutathione(GSH)/glutathione reductase (GR) system.

FIG. 3 illustrates several representative thioredthin-related pathwaysinvolved in cell proliferation and apoptosis. For thioredoxin (TX) topromote cell growth, inhibit apoptosis or stimulate cell proliferation,it must be in the reduced form. It should be noted, however, that TX isalso involved in many other intracellular pathways.

FIG. 4 illustrates the coupled thioredoxin (TX)/thioredoxin reductase(TXR) system.

FIG. 5 illustrates, in tabular form, the Primary Endpoint (i.e., themitigation or prevention of patient peripheral neuropathy) of the JapanPhase III Clinical Trial, as determined utilizing the PeripheralNeuropathy Questionnaire (PNQ©).

FIG. 6 illustrates, in tabular form, an evaluation of the statisticalpower observed in the Japan Phase III Clinical Trial with respect to thePrimary Endpoint (i.e., the mitigation or prevention of patientperipheral neuropathy), as measured by the Generalized EstimatingEquation (GEE) method.

FIG. 7 illustrates, in tabular form, a Secondary Endpoint (i.e., adecrease in patient hemoglobin, erythrocyte, and hematocrit levels) ofthe Japan Phase III Clinical Trial, in patient populations receivingTavocept™ (BNP7787) or placebo.

FIG. 8 illustrates, in tabular form, a Secondary Endpoint (i.e., tumorresponse rate to chemotherapy administration) of the Japan Phase IIIClinical Trial, in patient populations receiving either Tavocept™(BNP7787) or placebo, as measured by the physician or by the IndependentRadiological Committee (IRC) criteria.

FIG. 9 illustrates, in graphical form, a Secondary Endpoint (i.e.,patient survival) of the Japan Phase III Clinical Trial, in patientpopulations diagnosed with non-small cell lung carcinoma receivingeither Tavocept™ (BNP7787) or placebo.

FIG. 10 illustrates, in graphical form, a Secondary Endpoint (i.e.,patient survival) of the Japan Phase III Clinical Trial, in femalepatient populations receiving either Tavocept™ (BNP7787) or placebo.

FIG. 11 illustrates, in graphical form, a Secondary Endpoint (i.e.,patient survival) of the Japan Phase III Clinical Trial, in patientpopulations diagnosed with adenocarcinoma receiving either Tavocept™(BNP7787) or placebo.

FIG. 12 illustrates, in graphical form, the median patient survival(i.e., time to death in months) in the U.S. Phase II NSCLC ClinicalTrial, in patient populations diagnosed with non-small cell lungcarcinoma receiving chemotherapy with either Tavocept™ (BNP7787) or noTavocept™ treatment.

FIG. 13 illustrates, in tabular form, patient overall survival (OS) andpatient progression-free survival (PFS) in the U.S. Phase II NSCLCClinical Trial, in patient populations diagnosed with non-small celllung carcinoma receiving chemotherapy with either Tavocept™ (BNP7787) orno Tavocept™ treatment.

FIG. 14 illustrates, in graphical form, the median patient survival(i.e., time to death in months) in the U.S. Phase II NSCLC Phase IIClinical Trial, in patient populations diagnosed with adenocarcinomareceiving chemotherapy with either Tavocept™ (BNP7787) or no Tavocept™treatment.

FIG. 15 illustrates, in tabular form, the number of patientsexperiencing Grade 3 and Grade 4 treatment-related adverse events in theU.S. Phase II NSCLC Phase II Clinical Trial, in patient populationsdiagnosed with non-small cell lung carcinoma receiving chemotherapy witheither Tavocept™ (BNP7787) or no Tavocept™ treatment.

DETAILED DESCRIPTION OF THE INVENTION

The descriptions and embodiments set forth herein are not intended to beexhaustive, nor do they limit the present invention to the precise formsdisclosed. They are included to illustrate the principles of theinvention, and its application and practical use by those skilled in theart.

DEFINITIONS

As utilized herein, the term “generic structural formula” refers to thefixed structural part of the molecule of the formula given.

As utilized herein, the term “nucleophile” means an ion or molecule thatdonates a pair of electrons to an atomic nucleus to form a covalentbond; the nucleus that accepts the electrons is called an electrophile.This occurs, for example, in the formation of acids and bases accordingto the Lewis concept, as well as in covalent carbon bonding in organiccompounds.

As utilized herein the terms “fragments”, “moieties” or “substituentgroups” are the variable parts of the molecule, designated in theformula by variable symbols, such as R_(x), X or other symbols.Substituent Groups may consist of one or more of the following:

“C_(x)-C_(y) alkyl” generally means a straight or branched-chainaliphatic hydrocarbon containing as few as x and as many as y carbonatoms. Examples include “C₁-C₆ alkyl”, particularly “C₁-C₄ alkyl” (alsoreferred to as “lower alkyl”), which includes a straight or branchedchain hydrocarbon with no more than 6 total carbon atoms, and C₁-C₁₆alkyl, which includes a hydrocarbon with as few as one up to as many assixteen total carbon atoms, and the like. In the present application,the term “alkyl” is defined as comprising a straight or branched chainhydrocarbon of between 1 and 20 atoms, which can be saturated orunsaturated, and may include heteroatoms such as nitrogen, sulfur, andoxygen;

“C_(x)-C_(y) alkylene” means a bridging moiety formed of as few as “x”and as many as “y” —CH₂— groups. In the present invention, the term“alkylene” or “lower alkylene” is defined as comprising a bridginghydrocarbon having from 1 to 6 total carbon atoms which is bonded at itsterminal carbons to two other atoms (—CH₂—)_(x) where x is 1 to 6;

“C_(x)-C_(y) alkenyl or alkynyl” means a straight or branched chainhydrocarbon with at least one double bond (alkenyl) or triple bond(alkynyl) between two of the carbon atoms;

“Halogen” or “Halo” means chloro, fluoro, bromo or iodo;

“C_(x)-C_(y) Cycloalkyl” means a hydrocarbon ring or ring systemconsisting of one or more rings, fused or unfused, wherein at least oneof the ring bonds is completely saturated, with the ring(s) having fromx to y total carbon atoms;

“Acyl” means —C(O)—R, where R is hydrogen, C_(x)-C_(y) alkyl, aryl,C_(x)-C_(y) alkenyl, C_(x)-C_(y) alkynyl, and the like;

“Acyloxy” means —O—C(O)—R, where R is hydrogen, C_(x)-C_(y) alkyl, aryl,and the like;

“Aryl” generally means an aromatic ring or ring system consisting of oneor more rings, preferably one to three rings, fused or unfused, with thering atoms consisting entirely of carbon atoms. In the presentinvention, the term “aryl” is defined as comprising an aromatic ringsystem, either fused or unfused, preferably from one to three totalrings, with the ring elements consisting entirely of 5-8 carbon atoms;

“Arylalkyl” means an aryl moiety as defined above, bonded to thescaffold through an alkyl moiety (the attachment chain);

“Arylalkenyl” and “Arylalkynyl” mean the same as “Arylalkyl”, butincluding one or more double or triple bonds in the attachment chain;

“Amine” means a class of organic complexes of nitrogen that may beconsidered as derived from ammonia (NH₃) by replacing one or more of thehydrogen atoms with alkyl groups. The amine is primary, secondary ortertiary, depending upon whether one, two or three of the hydrogen atomsare replaced. A “short chain anime” is one in which the alkyl groupcontains from 1 to 10 carbon atoms;

“Ammine” means a coordination analog formed by the union of ammonia witha metallic substance in such a way that the nitrogen atoms are linkeddirectly to the metal. It should be noted the difference from amines, inwhich the nitrogen is attached directly to the carbon atom;

“Azide” means any group of complexes having the characteristic formulaR(N₃)_(x). R may be almost any metal atom, a hydrogen atom, a halogenatom, the ammonium radical, a complex [CO(NH₃)₆], [Hg(CN)₂M], (withM=Cu, Zn, Co, Ni) an organic radical like methyl, phenyl, nitrophenol,dinitrophenol, p-nitrobenzyl, ethyl nitrate, and the like. The azidegroup possesses a chain structure rather than a ring structure;

“Imine” means a class of nitrogen-containing complexes possessing acarbon-to-nitrogen double bond (i.e., R—CH═NH);

“Heterocycle” means a cyclic moiety of one or more rings, preferably oneto three rings, fused or unfused, wherein at least one atom of one ofthe rings is a non-carbon atom. Preferred heteroatoms include oxygen,nitrogen and sulfur, or any combination of two or more of those atoms.The term “Heterocycle” includes furanyl, pyranyl, thionyl, pyrrolyl,pyrrolidinyl, prolinyl, pyridinyl, pyrazolyl, imidazolyl, triazolyl,tetrazolyl, oxathiazolyl, dithiolyl, oxazolyl, isoxazolyl, oxadiazolyl,pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, oxazinyl, thiazolyl,and the like; and

“Substituted” modifies the identified fragments (moieties) by replacingany, some or all of the hydrogen atoms with a moiety (moieties) asidentified in the specification. Substitutions for hydrogen atoms toform substituted complexes include halo, alkyl, nitro, amino (alsoN-substituted, and N,N di-substituted amino), sulfonyl, hydroxy, alkoxy,phenyl, phenoxy, benzyl, benzoxy, benzoyl, and trifluoromethyl.

As utilized herein, the definitions for the terms “adverse event”(effect or experience), “adverse reaction”, and unexpected adversereaction have previously been agreed to by consensus of the more thanthirty Collaborating Centers of the WHO International Drug MonitoringCentre (Uppsala, Sweden). See, Edwards, I. R., et al., Harmonisation inPharmacovigilance Drug Safety 10(2):93-102 (1994). The followingdefinitions, with input from the WHO Collaborative Centre, have beenagreed to:

1. Adverse Event (Adverse Effect or Adverse Experience)—Any untowardmedical occurrence in a patient or clinical investigation subjectadministered a pharmaceutical product and which does not necessarilyhave to have a causal relationship with this treatment. An Adverse Event(AE) can therefore be any unfavorable and unintended sign (including anabnormal laboratory finding, for example), symptom, or diseasetemporally associated with the use of a medicinal product, whether ornot considered related to the medicinal product.

2. Adverse Drug Reaction (ADR)—In the pre-approval clinical experiencewith a new medicinal product or its new usages, particularly as thetherapeutic dose(s) may not be established: all noxious and unintendedresponses to a medicinal product related to any dose should beconsidered adverse drug reactions. Drug-related Adverse Events are ratedfrom grade 1 to grade 5 and relate to the severity or intensity of theevent. Grade 1 is mild, grade 2 is moderate, grade 3 is severe, grade 4is life threatening, and grade 5 results in death.

3. Unexpected Adverse Drug Reaction—An adverse reaction, the nature orseverity of which is not consistent with the applicable productinformation.

Serious Adverse Event or Adverse Drug Reaction: A Serious Adverse Event(experience or reaction) is any untoward medical occurrence that at anydose:(a) Results in death or is life-threatening. It should be noted that theterm “life-threatening” in the definition of “serious” refers to anevent in which the patient was at risk of death at the time of theevent; it does not refer to an event which hypothetically might havecaused death if it were more severe.(b) Requires inpatient hospitalization or prolongation of existinghospitalization.(c) Results in persistent or significant disability/incapacity, or(d) Is a congenital anomaly/birth defect.

As utilized herein the term “cancer” refers to all known forms of cancerincluding, solid forms of cancer (e.g., tumors), lymphomas, andleukemias.

As utilized herein, the term “clinical trial” or “trial”, refers to:

-   -   (i) the Japan Phase III Clinical Trial disclosed in the present        invention which was utilized to show the ability of Tavocept™        (also referred to in the literature as disodium        2,2′-dithio-bis-ethane sulfonate, dimesna, or BNP7787) to        prevent and/or reduce peripheral neuropathy induced by        paclitaxel/cisplatin combination therapy. The incidence and        severity of adverse reactions, time to their onset, etc. and the        like, were compared between patients treated with Tavocept™ and        those given a placebo using Quality of Life (QOL) questionnaires        (i.e., Peripheral Neuropathy Questionnaire (PNQ©) and CIPN-20))        and the National Cancer Institute-Common Toxicity Criteria        (NCI-CTC). The effects of Tavocept™ on the Quality of Life (QOL)        of patients under anticancer treatment were also evaluated using        the QOL questionnaire, EORTC QLQ-C30. Whether or not Tavocept™        would affect the efficacy of paclitaxel/cisplatin combination        therapy was also evaluated based on the response rate,        aggravation-free survival period, and total survival period. In        order to make all these evaluations, Tavocept™ (approximately        14-22 g/m², most preferably approximately 18.4 g/m²) or placebo        (0.9% NaCl) was administered to non-small cell lung carcinoma        (NSCLC) patients, including adenocarcinoma patients, under        chemotherapy with paclitaxel (approximately 160-190 mg/m², most        preferably approximately 175 mg/m²) and cisplatin (approximately        60-100 mg/m², most preferably approximately 80 mg/m²), every 3        weeks (and repeated for a minimum of 2 cycles); and/or    -   (ii) the United States (U.S.) Phase II non-small cell lung        carcinoma (NSCLC) Clinical Study disclosed in the present        invention was used to ascertain the effect of a dose-dense        administration of docetaxel and cisplatin every two weeks with        concomitant administration of pegfilgrastim and darbepoetin alfa        with and without administration of Tavocept™ (also referred to        in the literature as disodium 2,2′-dithio-bis-ethane sulfonate,        dimesna, or BNP7787) in patients with advanced stage (IIIB/IV)        non-small cell lung carcinoma (NSCLC), including adenocarcinoma        patients. Whether or not Tavocept™ would affect the efficacy of        the dose-dense docetaxel/cisplatin combination therapy was also        evaluated based on the response rate, aggravation-free survival        period, and total survival period. In order to make all these        evaluations, in the Tavocept™ arm of the clinical study,        docetaxel administration (75 mg/m²; i.v. administration over a        period of 1 hour on day one of the chemotherapy cycle) was        immediately followed by the administration of Tavocept™ (40 g;        i.v. administration over a period of 30 minutes). The Tavocept™        administration was then immediately followed by the        administration of cisplatin (75 mg/m²; i.v. administration over        a period of 1 hour) with adequate hydration. Darbepoetin alfa        (200 μg; subcutaneous administration) was administered on day        one of the chemotherapy cycle and pegfilgrastim (6 mg        subcutaneous administration) was administered on day two of the        chemotherapy cycle if the patient's hemoglobin levels were ≦11        g/dL. The aforementioned chemotherapy cycle was repeated every        two weeks, for up to a total of six cycles. The other,        non-Tavocept™ administration arm of the study was identical to        the previously discussed Tavocept™ arm, with the exception that        the docetaxel administration was immediately followed by        cisplatin administration without an intermediate administration        of Tavocept™. In addition, the incidence and severity of adverse        reactions were also compared between patients in the Tavocept™        and non-Tavocept™ arms of the study using the National Cancer        Institute-Common Toxicity Criteria (NCI-CTC) questionnaire.

As utilized herein, the term “adenocarcinoma” refers to a cancer thatoriginates in glandular tissue. Glandular tissue comprises organs thatsynthesize a substance for release such as hormones. Glands can bedivided into two general groups: (i) endocrine glands—glands thatsecrete their product directly onto a surface rather than through aduct, often into the blood stream and (ii) exocrine glands—glands thatsecrete their products via a duct, often into cavities inside the bodyor its outer surface. However, it should be noted that to be classifiedas adenocarcinoma, the tissues or cells do not necessarily need to bepart of a gland, as long as they have secretory properties.Adenocarcinoma may be derived from various tissues including, but notlimited to, breast, colon, lung, prostate, salivary gland, stomach,liver, gall bladder, pancreas (99% of pancreatic cancers are ductaladenocarcinomas), cervix, vagina, and uterus, as well as unknown primaryadenocarcinomas. Adenocarcinoma is a neoplasm which frequently presentsmarked difficulty in differentiating from where and from which type ofglandular tissue the tumor(s) arose. Thus, an adenocarcinoma identifiedin the lung may have had its origins (or may have metastasized) from anovarian adenocarcinoma. Cancer for which a primary site cannot be foundis called cancer of unknown primary.

As utilized herein, the term “non-small cell lung cancer (NSCLC)”accounts for approximately 75% of all primary lung cancers. NSCLC ispathologically characterized further into adenocarcinoma, squamous cellcarcinoma, large cell carcinoma, and various other less common forms.

As utilized herein, the terms “chemotherapy” or “chemotherapeuticregimen(s)” or “chemotherapy cycle” refer to treatment using theabove-mentioned chemotherapeutic agents with or without the use of anoxidative metabolism-affecting Formula (I) compound of the presentinvention.

As used herein, the term “potentiate”, “potentiating”, “chemotherapypotentiating”, “chemotherapeutic effect is potentiated”, and“potentiating the chemotherapeutic effects” is defined herein asproducing one or more of the following physiological effects: (i) theincrease or enhancement of the cytotoxic or cytostatic activity ofchemotherapy agents by acting in an additive or synergistic cytotoxicmanner with said chemotherapeutic agents within the tumor cells; (ii)reducing, preventing, mitigating, and/or delaying said deleteriousphysiological manifestations of said cancer in subjects sufferingtherewith; (iii) selectively sensitizing cancer cells to the anti-canceractivity of chemotherapeutic agents; and/or (iv) restoring apoptoticeffects or sensitivity in tumor cells.

As used herein, the term “chemotherapeutic agent” or “chemotherapyagent” or “chemotherapeutic drug” refer to an agent that reduces,prevents, mitigates, limits, and/or delays the growth of metastases orneoplasms, or kills neoplastic cells directly by necrosis or apoptosisof neoplasms or any other mechanism, or that can be otherwise used, in apharmaceutically-effective amount, to reduce, prevent, mitigate, limit,and/or delay the growth of metastases or neoplasms in a subject withneoplastic disease. Chemotherapeutic agents include, for example,fluoropyrimidines; pyrimidine nucleosides; purine nucleosides;anti-folates, platinum agents; anthracyclines/anthracenediones;epipodophyllotoxins; camptothecins; hormones; hormonal complexes;antihormonals; enzymes, proteins, peptides and polyclonal and/ormonoclonal antibodies; vinca alkaloids; taxanes; epothilones;antimicrotubule agents; alkylating agents; antimetabolites;topoisomerase inhibitors; aziridine-containing compounds; antivirals;and various other cytotoxic and cytostatic agents.

As utilized herein, the terms “chemotherapy”, “chemotherapeuticregimen(s)”, or “chemotherapy cycle” refer to treatment using theabove-mentioned chemotherapeutic agents with or without the Formula (I)compounds of the present invention.

As utilized herein, the term “chemotherapeutic effect” refers to theability of an agent to reduce, prevent, mitigate, limit, and/or delaythe growth of metastases or neoplasms, or kill neoplastic cells directlyby necrosis or apoptosis of neoplasms or any other mechanism, or thatcan be otherwise used to reduce, prevent, mitigate, limit, and/or delaythe growth of metastases or neoplasms in a subject with neoplasticdisease.

As utilized herein, the term “cycle” refers to the administration of acomplete regimen of medicaments to the patient in need thereof in adefined time period. By way of non-limiting example, in the Japan PhaseIII Clinical Trial disclosed herein, a cycle would comprise theadministration of taxane and platinum medicaments, an oxidativemetabolism-affecting Formula (I) compound, and any associatedmedications which may be required (e.g., pre-hydration, anti-emesisdrugs, and the like) to the patient within a defined time period.

As used herein, the term “cytostatic agents” are mechanism-based agentsthat slow the progression of neoplastic disease and include drugs,biological agents, and radiation.

As used herein the term “cytotoxic agents” are any agents or processesthat kill neoplastic cells and include drugs, biological agents, andradiation. In addition, the term “cytotoxic” is inclusive of the term“cytostatic”.

As used herein, the term “platinum medicaments” or “platinum compounds”include all compounds, compositions, and formulations which contain aplatinum ligand in the structure of the molecule. By way of non-limitingexample, the valence of the platinum ligand contained therein may beplatinum II or platinum IV. The platinum medicaments or platinumcompounds of the present invention include, in a non-limiting manner,cisplatin, oxaliplatin, carboplatin, satraplatin, and analogs andderivatives thereof.

As used herein, the term “taxane medicaments” include, in a non-limitingmanner, docetaxel or paclitaxel (including the commercially-availablepaclitaxel derivatives Taxol® and Abraxane®), polyglutamylated forms ofpaclitaxel (e.g., Xyotax®), liposomal paclitaxel (e.g., Tocosol®), andanalogs and derivatives thereof.

As utilized herein, the term “colony-stimulating factor” (CSF) aresecreted glycoproteins which bind to receptor proteins on the surfacesof hematopoietic stem cells and thereby activate intracellular signalingpathways which can cause the cells to proliferate and differentiate intoa specific kind of blood cell (usually white blood cells). Hematopoieticstem cells (HSC) are stem cells (i.e., cells retain the ability to renewthemselves through mitotic cell division and can differentiate into adiverse range of specialized cell types) that give rise to all the bloodcell types including myeloid (e.g., monocytes, macrophages,neutrophiles, basophils, eosinophils, erythrocytes,megakaryocytes/platelets, dendritic cells, and the like) and lymphoidlineages (e.g., T-cells, B-cells, NK-cells, and the like).Colony-stimulating factors include: macrophage colony-stimulating factor(CSF-1); granulocyte-macrophage colony-stimulating factor (CSF-2); andgranulocyte colony-stimulating factor (GCSF or CSF-3).

As used herein the term “erythropoiesis” refers to the process by whichred blood cells (erythrocytes) are produced. In the early fetus,erythropoiesis takes place in the mesodermal cells of the yolk sac. Bythe third or fourth month of fetal development, erythropoiesis moves tothe spleen and liver. In human adults, erythropoiesis generally occurswithin the bone marrow. The long bones of the arm (tibia) and leg(femur) cease to be important sites of hematopoiesis by approximatelyage 25; with the vertebrae, sternum, pelvis, and cranial bonescontinuing to produce red blood cells throughout life. However, itshould be noted that in humans with certain diseases and in someanimals, erythropoiesis also occurs outside the bone marrow, within thespleen or liver. This is termed extramedullary erythropoiesis. In theprocess of red blood cell maturation, a cell undergoes a series ofdifferentiations. The following stages of development all occur withinthe bone marrow: (i) pluripotent hematopoietic stem cell; (ii)multipotent stem cell; (iii) unipotent stem cell; (iv) pronormoblast;(v) basophilic normoblast/early normoblast; (vi) polychrmatophilicnormoblast/intermediate normoblast; (vii) orthochromic normoblast/latenormoblast; and (viii) reticulocyte. Following these stages, the cell isreleased from the bone marrow, and ultimately becomes an “erythrocyte”or mature red blood cell circulating in the peripheral blood.

As used herein, the term “erythropoietin” is a glycoprotein hormone thatis a cytokine for erythrocyte (red blood cell) precursors in the bonemarrow which regulates the process of red blood cell production (i.e.,erythropoiesis). Erythropoietin (EPO) is produced mainly by peritubularfibroblasts of the renal cortex. Regulation is believed to rely on afeed-back mechanism measuring blood oxygenation. Constitutivelysynthesized transcription factors for EPO, known as hypoxia induciblefactors (HIFs), are hydroxylized and proteosomally-digested in thepresence of oxygen.

As used herein, the term “darbepoetin alfa” is an synthetic form oferythropoietin. It is an erythropoiesis stimulating (i.e., increases redblood cell levels) protein, comprised of 165-amino acid residues, and isused to treat anemia, commonly associated with chronic renal failure andcancer chemotherapy. Darbepoetin is marketed by Amgen under the tradename Aranesp. It is produced by recombinant DNA technology in modifiedChinese hamster ovary cells. It differs from endogenous erythropoietinby containing two more N-linked oligosaccharide chains.

As utilized herein, the term “pegfilgrastim” is an immunostimulatorwhich functions as a pegylated granulocyte colony-stimulating factor(GCSF). Amgen manufactures pegfilgrastim under the brand name Neulasta.GCSF is a colony-stimulating factor hormone. It is a glycoprotein,growth factor or cytokine produced by endothelium, macrophages, and anumber of other immune cells, which stimulates the bone marrow toproduce granulocytes and stem cells. GCSF then stimulates the bonemarrow to release them into the blood. It also stimulates the survival,proliferation, differentiation, and function of neutrophil precursorsand mature neutrophils. GCSF is also known as colony-stimulating factor3 (CSF 3). The natural human glycoprotein exists in two forms; a 174-and 180-amino acid residue protein with a molecular weight of 19.6 kDa.The more-abundant and more-active 174 amino acid residue form has beenused in the development of pharmaceutical products by recombinant DNA(rDNA) technology. Pegylation is the process of covalent attachment ofpolyethylene glycol (PEG) polymer chains to another molecule, normally adrug or therapeutic protein. Pegylation is routinely achieved byincubation of a reactive derivative of PEG with the targetmacromolecule. The covalent attachment of PEG to a drug or therapeuticprotein can facilitate the “masking” of the agent from the host's immunesystem (i.e., causing reduced immunogenicity and antigenicity) andincrease the hydrodynamic size (i.e., size in solution) of the agentwhich prolongs its circulatory time by reducing renal clearance.Pegylation can also provide water solubility to hydrophobic drugs andproteins.

As used herein, the term “evidence of” as it applies to the exhibitionof thioredoxin-mediated or glutaredoxin-mediated treatment resistance inthe present invention means that it is probable or likely thatthioredoxin-mediated or glutaredoxin-mediated treatment resistance hasoccurred or will occur. It is described in that manner due to the factthat it is neither expected, nor possible to prove with 100% certaintythat the cancer cells exhibit thioredoxin-mediated orglutaredoxin-mediated treatment resistance, prior to the actualtreatment of the patient. By way of non-limiting example, the currentuse of, e.g., florescence in situ hybridization (FISH) orimmunohistochemistry (IHC) to guide treatment decisions forHER2/neu-based therapy are predicated upon the probability of theoverexpression/increased concentrations of HER2/neu being correlatedwith the probability of a therapeutic response. Such expectation of atherapeutic response is not 100% certain, and is related to manyfactors, not the least of which is the diagnostic accuracy of the testutilized which, in turn, is also limited by the sampling of the tumorand various other factors (e.g., laboratory methodology/technique,reagent quality, and the like).

As used herein, the terms “Formula (I) compound” or “Formula (I)composition” include all molecules, unless specifically identifiedotherwise, that share substantial structural and/or functionalcharacteristics with the 2,2′-dithio-bis-ethane sulfonate parentcompound and includes the compounds of Formula (I) which refers tocompounds possessing the generic structural formula:

X—S—S—R₁-R₂:

wherein;

R₁ is a lower alkylene, wherein R₁ is optionally substituted by a memberof the group comprising: lower alkyl, aryl, hydroxy, alkoxy, aryloxy,mercapto, alkylthio or arylthio, for a corresponding hydrogen atom, or

-   -   R₂ and R₄ is sulfonate or phosphonate;    -   R₅ is hydrogen, hydroxy, or sulfhydryl;    -   m is 0, 1, 2, 3, 4, 5, or 6; and    -   X is a sulfur-containing amino acid or a peptide comprising from        2-10 amino acids;    -   or wherein X is a member of the group comprising a: lower        thioalkyl (lower mercapto alkyl), lower alkylsulfonate, lower        alkylphosphonate, lower alkenylsulfonate, lower alkyl, lower        alkenyl, lower alkynyl, aryl, alkoxy, aryloxy, mercapto,        alkylthio or hydroxy for a corresponding hydrogen atom.        The Formula (I) compounds or compositions of the present        invention also include pharmaceutically-acceptable salts,        prodrugs, analogs, conjugates, hydrates, solvates, polymorphs,        stereoisomers (including diastereoisomers and enantiomers) and        tautomers thereof.        By way of non-limiting example, the Formula (I) compounds or        compositions of the present invention include the disodium salt        of 2,2′-dithio-bis-ethane sulfonate (which has also been        referred to in the literature as dimesna, Tavocept™, and        BNP7787). Additionally, by way of non-limiting example, the        Formula (I) compounds or compositions of the present invention        include the metabolite of disodium 2,2′-dithio-bis-ethane        sulfonate, known as 2-mercapto ethane sulfonate sodium (also        known in the literature as mesna) or 2-mercapto ethane sulfonate        conjugated with a substituent group consisting of:

-Cys, -Homocysteine, -Cys-Gly, -Cys-Glu, -Homocysteine,-Homocysteine-Gly, -Homocysteine-Glu, -Cys-Glu,

wherein R₁ and R₂ are any L- or D-amino acids.

It should be noted that all of the aforementioned chemical entities andcompounds in the previous two (2) paragraphs are included in Formula (I)compounds of the present invention. The compounds of Formula (I) includepharmaceutically-acceptable salts of such compounds, as well asprodrugs, analogs, conjugates, hydrates, solvates and polymorphs, aswell as stereoisomers (including diastereoisomers and enantiomers) andtautomers of such compounds. Compounds of Formula (I), and theirsynthesis are described in, e.g., U.S. Pat. Nos. 5,808,160, 5,922,902,6,160,167, and 6,504,049; and Published U.S. Patent Application No.2005/0256055, the disclosures of which are hereby incorporated byreference in their entirety.

As used herein, the terms “heteroconjugates”, “mesna heteroconjugate”,“mesna conjugate”, or “mesna derivative” represent the metabolite ofdisodium 2,2′-dithio-bis-ethane sulfonate, known as 2-mercapto ethanesulfonate sodium (mesna), as a disulfide form which is conjugated with asubstituent group consisting of:

-Cys, -Homocysteine, -Cys-Gly, -Cys-Glu, -Homocysteine,-Homocysteine-Gly, -Homocysteine-Glu, -Cys-Glu,

wherein R₁ and R₂ are any L- or D-amino acids. Mesna heteroconjugatecompounds are included in the Formula (I) compounds and may besynthesized as described in Published U.S. Patent Application2005/0256055, the disclosure of which is incorporated herein, byreference, in its entirety.

As utilized herein, the term “oxidative metabolism-affecting compound”is a compound, formulation, or agent which is capable of: mitigating orpreventing: (i) the overexpression (or increased activity, or both) ofthioredoxin or glutaredoxin in cancer cells; (ii) the loss of apoptoticsensitivity to therapy (i.e., drug or ionizing radiation resistance);(iii) increased conversion of RNA into DNA (involving ribonucleotidereductase); (iv) altered gene expression; (v) increased cellularproliferation signals and rates; (vi) increased thioredoxin peroxidase;and/or (vii) increased angiogenic activity (i.e., increased blood supplyto the tumor). Accordingly, by pharmacological inactivation ormodulation of thioredoxin and/or glutaredoxin by the proper medicaladministration of effective levels and schedules of the oxidativemetabolism-affecting compounds of the present invention, can result inenhancement of chemotherapy effects and thereby lead to increasedpatient survival.

As used herein, a “medically-sufficient dose” or a “medically-sufficientamount” in reference to the compounds or compositions of the instantinvention refers to the dosage that is sufficient to induce a desiredbiological, pharmacological, or therapeutic outcome in a subject withneoplastic disease. That result can be: (i) cure or remission ofpreviously observed cancer(s); (ii) shrinkage of tumor size; (iii)reduction in the number of tumors; (iv) delay or prevention in thegrowth or reappearance of cancer; (v) selectively sensitizing cancercells to the anti-cancer activity of chemotherapeutic agents; (vi)restoring or increasing apoptotic effects or sensitivity in tumor cells;and/or (vii) increasing the time of survival of the patient, alone orwhile concurrently experiencing reduction, prevention, mitigation,delay, shortening the time to resolution of, alleviation of the signs orsymptoms of the incidence or occurrence of an expected side-effect(s),toxicity, disorder or condition, or any other untoward alteration in thepatient.

As used herein, the term “g/m²” represents the amount of a givencompound or formulation in grams per square meter of the total bodysurface area of the subject to whom the compound or formulation isadministered.

As used herein, the term “mg/m²” represents the amount of a givencompound or formulation in milligrams per square meter of the total bodysurface area of the subject to whom the compound or formulation isadministered.

As utilized herein, the term “patient” refers to any individual orsubject, without limitation, who is in need of treatment with acompound, composition, medicament, formulation, method, or kit which isdisclosed in the present invention.

As used herein, the term “pre-treatment” comprises the administration ofone or more medications, said administration occurring at any time priorchemotherapy administration in accordance with both the methods knownwithin the art and the patient's medical condition.

As used herein, the term “pharmaceutically-acceptable salt” means saltderivatives of drugs which are accepted as safe for humanadministration. In the present invention, the Formula (I) compounds ofthe present invention include pharmaceutically-acceptable salts, whichinclude but are not limited to: (i) a monosodium salt; (ii) a disodiumsalt; (iii) a sodium potassium salt; (iv) a dipotassium salt; (v) acalcium salt; (vi) a magnesium salt; (vii) a manganese salt; (viii) anammonium salt; and (ix) a monopotassium salt.

As used herein the term “Quality of Life” or “QOL” refers, in anon-limiting manner, to a maintenance or increase in a cancer patient'soverall physical and mental state (e.g., cognitive ability, ability tocommunicate and interact with others, decreased dependence uponanalgesics for pain control, maintenance of ambulatory ability,maintenance of appetite and body weight (lack of cachexia), lack of ordiminished feeling of “hopelessness”; continued interest in playing arole in their treatment, and other similar mental and physical states).

As used herein the terms “reactive oxygen species (ROS)” and “reactivenitrogen species (RNS)” refer to ionic species which may result from avariety of metabolic and/or environmental processes. By way ofnon-limiting example, intracellular ROS (e.g., hydrogen peroxide: H₂O₂,superoxide anion: O₂ ⁻, hydroxyl radical: OH⁻, nitric oxide, and thelike) may be generated by several mechanisms: (i) by the activity ofradiation; (ii) during xenobiotic and drug metabolism; and (iii) underrelative hypoxic, ischemic and catabolic metabolic conditions.

As used herein, the term “reducing” includes preventing, attenuating theoverall severity of, delaying the initial onset of, and/or expeditingthe resolution of the acute and/or chronic pathophysiology associatedwith malignancy in a subject.

As used herein the term “redox state”, “redox potential”,“oxidative/reductive state” of any particular biological environment canbe defined as the sum of oxidative and reductive processes occurringwithin that environment, which affects the extent to which molecules areoxidized or reduced within it. The redox potential of biological ions ormolecules is a measure of their tendency to lose an electron (i.e.,thereby becoming oxidized). Under normal physiological circumstances,most intracellular biological systems are predominantly found in areduced state. Within cells, thiols (R—SH) such as glutathione (GSH) aremaintained in their reduced state, as are the nicotinamide nucleotidecoenzymes NADH and NADPH. Conversely, plasma is generally an oxidizingenvironment due to the high partial pressure of oxygen and the relativeabsence of disulfide reducing enzymes. Physiological circumstances can,however, arise which alter the overall redox balance and lead to a moreoxidizing environment on cells. In biological systems, this activityarises as a result of changes in intracellular oxidative metabolism andphysiological systems have evolved to preserve, protect, and control thenormal reducing environment. However, when the changes overwhelm theseprotective mechanisms, oxidative damage and profound biological changescan occur. Cancer cells have been observed to have the ability to mountmore effective anti-oxidative responses to changes in intracellularoxidative metabolism (e.g., oxidative stress) in comparison to normal,non-cancerous, cells, thereby leading to a survival advantage and theability to resist or escape the anti-cancer and cytotoxic action ofchemotherapeutic agent(s).

As utilized herein, the term “redox response” refers to the biologicalresponse to induce antioxidant systems against changes in oxidativemetabolism to maintain the homeostasis in the intracellular redoxbalance.

As used herein, the term “receive” or “received” refers to a subject whohas cancer and who has received, is currently receiving, or will receiveone or more chemotherapeutic agents and/or an oxidativemetabolism-affecting Formula (I) compound of the present invention.

As used herein the term “synergism” or “synergistic” means theanti-cancer activity achieved by the above-defined Formula (I) compoundsin combination with chemotherapeutic agent(s) is greater than theanti-cancer activity achieved by either form of treatment individually.For example, this may be mathematically expressed as the synergisticresult of treatment with Drugs A+B administered together (as taughtherein)=Result C>Drug A Result, alone+Drug B Result, alone. In contrast,a purely additive result may be mathematically expressed as: Drugs A+Badministered together=Result C=Drug A Result, alone+Drug B Result,alone. In the foregoing examples, Drug A can represent Formula (I)compounds and the observed treatment result alone or combined, and DrugB can represent any single chemotherapy agent or combination ofchemotherapy agents that are administered alone.

The term “solvate” or “solvates” refers to a molecular complex of acompound such as an oxidative metabolism-affecting Formula (I) compoundof the present invention with one or more solvent molecules. Suchsolvent molecules are those commonly used in the pharmaceutical art(e.g., water, ethanol, and the like). The term “hydrate” refers to thecomplex where the solvent molecule is water.

As used herein, the term “treat” or “treated”, with respect to a patientwithout cancer, refers to a patient, who is in need thereof, and who hasreceived, is currently receiving, or will receive Formula (I) compoundsof the present invention.

As used herein, the term “treat” or “treated”, with respect to a patientwith cancer, refers to a patient who has received, is currentlyreceiving, or will receive one or more chemotherapeutic agents and/orFormula (I) compounds of the present invention.

As used herein, “treatment schedule time” means the difference inschedule of administration time, including: (i) the amount of drugadministered per day or week; (ii) the amount of drug administered perday or week per m² of body surface area; and (iii) the amount of drugadministered per day or week per kg of body weight.

As used herein, “difference in administration of drug treatment time”,means permitting administration of treatment to occur in materially lesstime (a reduction in time from, e.g., 4 hours to 1 hour, from one day to6 hours, and the like) thereby allowing the patient to minimize time inthe outpatient or hospitalized treatment time.

As used herein, “treatment schedule time” or “treatment regimen” meansthe difference in schedule of administration time, including: (i) theamount of drug administered per day or week; (ii) the amount of drugadministered per day or week per m² of body surface area; or (iii) theamount of drug administered per day or week per kg of body weight.

Many types of cancer cells have been shown to have increased expressionand/or activity of thioredoxin and/or glutaredoxin including, but notlimited to, lung cancer, colorectal cancer, gastric cancer, esophagealcancer, ovarian cancer, cancer of the biliary tract, gallbladder cancer,cervical cancer, breast cancer, endometrial cancer, vaginal cancer,prostate cancer, uterine cancer, hepatic cancer, pancreatic cancer, andadenocarcinoma. The overexpression (and possibly increased activity) ofthioredoxin and/or glutaredoxin in cancer cells results in chemotherapydrug resistance to apoptosis. Such overexpression leads, e.g., toshortened patient survival that is believed to be mediated by increasedconcentrations or expression of thioredoxin/glutaredoxin, which in turnpromote tumor-mediated resistance to chemotherapy-induced apoptosis,overexpression of oxidoperoxidases, increased conversion of RNA intoDNA, increased nuclear transcription, increased cell proliferation,and/or increased angiogenesis, any of which can act in concert toprovide the cancer cells the ability to resist chemotherapy andradiation therapy.

The present invention involves the medicinal and pharmacologicalinactivation and modulation of the thioredoxin/glutaredoxin system whichthereby inactivates, reverses or modulates the drug-resistant propertiesin the cancer cells that are otherwise imparted by the increased levelsor overexpression of thioredoxin/glutaredoxin in said cancer cells. Themedicinal and pharmacological inactivation involves the administrationof an oxidative metabolism-affecting Formula (I) compound of the presentinvention. Any of the aforementioned types of cancer that have increasedexpression or concentrations of thioredoxin and/or glutaredoxin aresusceptible to and may benefit from thioredoxin-/glutaredoxin-basedintervention by the present invention. The present invention alsoteaches how to optimize the schedule, dose, and combination ofchemotherapy regimens in patients by the identification in-advance ofand through-out treatment of the thioredoxin/glutaredoxin levels and themetabolic state within a sample of cancer cells isolated from theindividual patients. Moreover, the use of kits that enable diagnosticand therapeutic optimization of the compositions and methods of thepresent invention to further enhance the survival outcome and benefit topatients by, for example, the determination of the optimumchemotherapeutic drug regimen to utilize. The present invention alsoteaches how to identify patients, in advance, who would not be likely tobenefit from such intervention by the use of diagnostic kits, therebyallowing other treatment approaches that may be more clinicallyefficacious to be pursued.

I. Glutathione and Cysteine

Glutathione (GSH), a tripeptide (γ-glutamyl-cysteinyl-glycine) serves ahighly important role in both intracellular and extracellular redoxbalance. It is the main derivative of cysteine, and the most abundantintracellular non-protein thiol, with an intracellular concentrationapproximately 10-times higher than other intracellular thiols. Withinthe intracellular environment, glutathione (GSH) is maintained in thereduced form by the action of glutathione reductase and NADPH. Underconditions of oxidative stress, however, the concentration of GSHbecomes markedly depleted. Glutathione functions in many diverse rolesincluding, but not limited to, regulating antioxidant defenses,detoxification of drugs and xenobiotics, and in the redox regulation ofsignal transduction. As an antioxidant, glutathione may serve toscavenge intracellular free radicals directly, or act as a co-factor forvarious other protection enzymes. In addition, glutathione may also haveroles in the regulation of immune response, control of cellularproliferation, and prostaglandin metabolism. Glutathione is alsoparticularly relevant to oncology treatment because of its recognizedroles in tumor-mediated drug resistance to chemotherapeutic agents andionizing radiation. Glutathione is able to conjugate electrophilic drugssuch as alkylating agents and cisplatin under the action of glutathioneS-transferases. Recently, GSH has also been linked to the efflux ofother classes of agents such as anthracyclines via the action of themultidrug resistance-associated protein (MRP). In addition to drugdetoxification, GSH enhances cell survival by functioning in antioxidantpathways that reduce reactive oxygen species, and maintain cellularthiols (also known as non-protein suifhydryis (NPSH)) in their reducedstates. See, e.g., Kigawa J, et al., Gamma-glutamyl cysteine synthetaseup-regulates glutathione and multidrug resistance-associated protein inpatients with chemoresistant epithelial ovarian cancer. Clin. CancerRes. 4:1737-1741 (1998).

Cysteine, another important NPSH, as well as glutathione are also ableto prevent DNA damage by radicals produced by ionizing radiation orchemical agents. Cysteine concentrations are typically much lower thanGSH when cells are grown in tissue culture, and the role of cysteine asan in vivo cytoprotector is less well-characterized. However, on a molarbasis cysteine has been found to exhibit greater protective activity onDNA from the side-effect(s) of radiation or chemical agents.Furthermore, there is evidence that cysteine concentrations in tumortissues can be significantly greater than those typically found intissue culture.

A number of studies have examined GSH levels in a variety of solid humantumors, often linking these to clinical outcome See, e.g., Hochwald, S.N., et al., Elevation of glutathione and related enzyme activities inhigh-grade and metastatic extremity soft tissue sarcoma. American Surg.Oncol. 4:303-309 (1997); Ghazal-Aswad, S., et al., The relationshipbetween tumour glutathione concentration, glutathione S-transferaseisoenzyme expression and response to single agent carboplatin inepithelial ovarian cancer patients. Br. J. Cancer 74:468-473 (1996);Berger, S. J., et al., Sensitive enzymatic cycling assay forglutathione: Measurement of glutathione content and its modulation bybuthionine sulfoximine in vivo and in vitro human colon cancer. CancerRes. 54:4077-4083 (1994). Wide ranges of tumor GSH concentrations havebeen reported, and in general these have been greater (i.e., up to10-fold) in tumors compared to adjacent normal tissues. Most researchershave assessed the GSH content of bulk tumor tissue using enzymaticassays, or GSH plus cysteine using HPLC.

In addition, cellular thiols/non-protein sulfhydryls (NPSH), e.g.,glutathione, have also been associated with increased tumor resistanceto therapy by mechanisms that include, but are not limited to: (i)conjugation and excretion of chemotherapeutic agents; (ii) direct andindirect scavenging of reactive oxygen species (ROS) and reactivenitrogen species (RNS); and (iii) maintenance of the “normal”intracellular redox state. Low levels of intracellular oxygen withintumor cells (i.e., tumor hypoxia) caused by aberrant structure andfunction of the associated tumor vasculature, has also been shown to beassociated with chemotherapy therapy-resistance andbiologically-aggressive malignant disease. Oxidative stress, commonlyfound in regions of intermittent hypoxia, has been implicated inregulation of glutathione metabolism, thus linking increased NPSH levelsto tumor hypoxia. Therefore, it is also important to characterize bothNPSH expression and its relationship to tumor hypoxia in tumors andother neoplastic tissues.

The heterogeneity of NPSH levels was examined in multiple biopsiesobtained from patients with cervical carcinomas who were entered into astudy investigating the activity of cellular oxidation and reductionlevels (specifically, hypoxia) on the response to radical radiotherapy.See, e.g., Fyles, A., et al., (Oxygenation predicts radiation responseand survival in patients with cervix cancer. Radiother. Oncol.48:149-156 (1998). The major findings from this study were that theintertumoral heterogeneity of the concentrations of GSH and cysteineexceeds the intratumoral heterogeneity, and that cysteine concentrationsof approximately 21 mM were found in some samples, confirming an earlierreport by Guichard, et al., (Glutathione and cysteine levels in humantumour biopsies. Br. J. Radiol. 134:63557-635561 (1990)). These levelsof cysteine are much greater than those typically seen in tissueculture, suggesting that cysteine might exert a significantradioprotective activity in cervical carcinomas and possibly other typesof cancer.

There is also extensive literature showing that elevated cellularglutathione levels can produce drug resistance in experimental models,due to drug detoxification or to the antioxidant activity of GSH. Inaddition, radiation-induced DNA radicals can be repairednon-enzymatically by GSH and cysteine, indicating a potential role forNPSH in radiation resistance. While cysteine is the more effectiveradioprotective agent, it is usually present in lower concentrationsthan GSH. Interestingly, under fully aerobic conditions, thisradioprotective activity appears to be relatively minor, and NPSHcompete more effectively with oxygen for DNA radicals under the hypoxicconditions that exist in some solid tumors, which might play asignificant role in radiation resistance.

Radiotherapy has traditionally been a major treatment modality forcervical carcinomas. Randomized clinical trials (Rose, D., et al.,Concurrent cisplatin-based radiotherapy and chemotherapy for locallyadvanced cervical cancer. New Engl. J. Med. 340:1144-1153 (1999)) showthat patient outcome is significantly improved when radiation therapy iscombined with cisplatin-based chemotherapy, and combined modalitytherapy is now widely being utilized in treatment regimens. It isimportant to establish the clinical relevance of GSH and cysteine levelsto drug and radiation resistance because of the potential to modulatethese levels using agents such as buthionine sulfoximine; anirreversible inhibitor of γ-glutanylcysteine synthetase that can produceprofound depletion of GSH in both tumor and normal tissues. See, e.g.,Bailey, T., et al., Phase I clinical trial of intravenous buthioninesulfoximine and melphalan: An attempt at modulation of glutathione. J.Clin. Oncol. 12:194-205 (1994). Evaluation of GSH concentrations havereported elevated tumor GSH relative to adjacent normal tissue, andintertumoral heterogeneity in GSH content. These findings are consistentwith the idea that GSH could play a clinically significant role in drugresistance. although it should be noted that relatively few studies havethe sample size and follow up duration necessary to detect a significantrelation between tumor GSH content and response to chemotherapy, hencethere are no consistent clinical data to support this idea.

Koch and Evans (Cysteine concentrations in rodent tumors: unexpectedlyhigh values may cause therapy resistance. Int. J. Cancer 67:661-667(1996)) have shown that cysteine concentrations in established tumorcell lines can be much greater when these are grown as in vivo tumors,as compared to the in vitro values, suggesting that cysteine might playa more significant role in therapy resistance than previouslyconsidered. Although relatively few studies have reported on cysteinelevels in human cancers, an earlier HPLC-based study of cervicalcarcinomas by Guichard, D. G., et al., (Glutathione and cysteine levelsin human tumour biopsies. Br. J. Radiol. 134:63557-635561 (1990)reported cysteine concentrations greater than 1 mM in a significantnumber of cases. Thus, the fact that the variability in cysteine levelsis greater than that for GSH suggests that these two thiols areregulated differently in tumors. By way of non-limiting example, theinhibition of γ-glutamylcysteine synthetase with the intravenousadministration of buthionine sulfoximine (BSO) could result in elevatedcellular levels of cysteine, due to the fact that the γ-glutamylcysteinesynthetase is not being utilized for GSH de novo synthesis. Similar toGSH, cysteine possesses the ability to repair radiation-induced DNAradicals and cysteine also has the potential to detoxify cisplatin; acytotoxic agent now routinely combined with radiotherapy to treatlocally-advanced cervical carcinomas.

II. Glutaredoxin

Glutaredoxin and thioredoxin (TX) are members of the thioredoxinsuperfamily; that mediate disulfide exchange via their Cys-containingcatalytic sites. While glutaredoxins mostly reduce mixed disulfidescontaining glutathione, thioredoxins are involved in the maintenance ofprotein sulfhydryls in their reduced state via disulfide bond reduction.See, e.g., Print, W. A., et al., The role of the thioredoxin andglutaredoxin pathways in reducing protein disulfide bonds in theEscherichia coli cytoplasm. J. Biol. Chem. 272:15661-15667 (1996). Thereduced form of thioredoxin is generated by the action of thioredoxinreductase; whereas glutathione provides directly the reducing potentialfor regeneration of the reduced form of glutaredoxin.

Glutaredoxins are small redox enzymes of approximately 100 amino acidresidues, which use glutathione as a cofactor. Glutaredoxins areoxidized by substrates, and reduced non-enzymatically by glutathione. Incontrast to thioredoxins, which are reduced by thioredoxin reductase, nooxidoreductase, other than described in the present invention, existsthat specifically reduces glutaredoxins. Instead, oxidized glutathioneis regenerated by glutathione reductase. Together these componentscomprise the glutathione system. See, e.g., Holmgren, A. and Fernandes,A. P., Glutaredoxins: glutathione-dependent redox enzymes with functionsfar beyond a simple thioredoxin backup system. Antioxid. Redox. Signal.6:63-74 (2004); Holmgren, A., Thioredoxin and glutaredoxin systems. J.Biol. Chem. 264:13963-13966 (1989).

Glutaredoxins basically function as electron carriers in theglutathione-dependent synthesis of deoxyribonucleotides by the enzymeribonucleotide reductase. Like thioredoxin, which functions in a similarway, glutaredoxin possesses an active catalytic site disulfide bond. Itexists in either a reduced or an oxidized form where the two cysteineresidues are linked in an intramolecular disulfide bond. Human proteinscontaining this domain include: glutaredoxin thioltransferase (GLRX);glutaredoxin 2 (GLRX2); thioredoxin-like 2 (GLRX3); GLRX5; PTGES2; andTXNL3. See, e.g., Nilsson, L. and Foloppe, N., The glutaredoxin-C-P-Y-C- motif: influence of peripheral residues. Structure 12:289-300(2004).

At least two glutaredoxin proteins exist in mammalian cells (12 or 16kDa), and glutaredoxin, like thioredoxin, cycles between disulfide anddithiol forms. The conversion of glutaredoxin from the disulfide form(oxidized) to the dithiol (reduced) form is catalyzed non-enzymaticallyby glutathione and is illustrated, below. In turn, glutathione cyclesbetween a thiol form (glutathione) that can reduce glutaredoxin and adisulfide form (glutathione disulfide); glutathione reductaseenzymatically reduces glutathione disulfide to glutathione. Thisreaction is illustrated below:

While the -CysXaaXaaCys- intramolecular disulfide bond is an essentialpart of the catalytic cycle for thioredoxin and protein disulfideisomerase, the most important oxidized species for glutaredoxins is aglutathionylated form as shown in Panel B.

III. The Thioredoxin Reductase (TRX)/Thioredoxin (TX) System ThioredoxinReductase (TRX)

The thioredoxin system is comprised of thioredoxin reductase (TXR) andits main protein substrate, thioredoxin (TX), where the catalytic sitedisulfide of TX is reduced to a dithiol by TXR at the expense of NADPH.The thioredoxin system, together with the glutathione system (comprisingNADPH, the flavoprotein glutathione reductase, glutathione, andglutaredoxin), is regarded as a main regulator of the intracellularredox environment, exercising control of the cellular redox state andantioxidant defense, as well as governing the redox regulation ofseveral cellular processes. The system is involved in direct regulationof: (i) several transcription factors, (ii) apoptosis (i.e., programmedcell death) induction, and (iii) many metabolic pathways (e.g., DNAsynthesis, glucose metabolism, selenium metabolism, and vitamin Crecycling). See, e.g., Amér, E. S. J., et al., Physiological functionsof thioredoxin and thioredoxin reductase. Eur. J. Biochem. 267:6102-6109(2000). In addition to TXs, other endogenous substrates have beendemonstrated for TXRs including, but not limited to, lipoic acid; lipidhydroperoxides; the cytotoxic peptide NK-lysin; vitamin K;dehydroascorbic acid; the ascorbyl free radical; and thetumor-suppressor protein p53. See, e.g., Reed, D. J., Molecular andCellular Mechanisms of Toxicity (DeMatteis, F. and Smith, L. L., eds.),pp. 35-68, CRC Press, Boca Raton (2002). However, the exactphysiological role that TXRs play in the reduction of most of thesesubstrates has not yet been fully defined.

The mammalian thioredoxin reductases (TXRs) are enzymes belonging to theavoprotein family of pyridine nucleotide-disulfide oxidoreductases thatincludes lipoamide dehydrogenase, glutathione reductase, and mercuricion reductase. Members of this family are homodimeric proteins in whicheach monomer includes an FAD prosthetic group, an NADPH binding site andan active site containing a redox-active disulfide. Electrons aretransferred from NADPH via FAD to the active-site disulfide of TXR,which then reduces the substrate. See, e.g., Williams, C. H., Chemistryand Biochemistry of Flavoenzymes (Muller, F., ed.), pp. 121-211, CRCPress, Boca Raton (1995).

TXRs are named for their ability to reduce oxidized thioredoxins (TXs),a group of small, ubiquitous redox-active peptides that undergoesreversible oxidation/reduction of two conserved cysteine (Cys) residueswithin the catalytic site. The mammalian TXRs are selenium-containingflavoproteins that possess: (i) a conserved -Cys-Val-Asn-Val-Gly-Cys-catalytic site; (ii) an NADPH binding site; and (iii) a C-terminalCys-Selenocysteine sequence that communicates with the catalytic siteand is essential for its redox activity. See, e.g., Powis, G. andMonofort, W. R. Properties and biological activities of thioredoxins.Ann. Rev. Pharmacol. Toxicol. 41:261-295 (2001). These proteins exist ashomodimers and undergo reversible oxidation/reduction. The activity ofTXR is regulated by NADPH, which in turn is produced byglucose-6-phosphate dehydrogenase (G6DP), the rate-limiting enzyme ofthe oxidative hexose monophosphate shunt (HMPS; also known as thepentose phosphate pathway). Two human TXR isozyme genes have beencloned: a 54 Kda enzyme that is found predominantly in the cytoplasm(TXR-1) and a 56 Kda enzyme that contains a mitochondrial importsequence (TXR-2). Id. A third isoform of TXR, designated (TGR) is a TXand glutathione reductase localized mainly in the testis, has also beenidentified. See, e.g., Sun, Q. A., et al., Selenoprotein oxidoreductasewith specificity for thioredoxin and glutathione systems. Proc. Natl.Acad. Sci. USA 98:3673-3678 (2001). Additionally, both mammaliancytosolic TX-1 and mitochondrial TX-2 have alternative splice variants.In humans, five different 5′ cDNA variants have been reported. One ofthe splicing variants exhibits a 67 kDa protein with an N-terminalelongation instead of the common 55 kDa. The physiological functions ofthese TXR splice variants have yet to be elucidated. See, e.g., Sun, Q.A., et al., Heterogeneity within mammalian thioredoxin reductases:evidence for alternative exon splicing. J. Biol. Chem. 276:3106-3114(2001).

The TXR-1 isozyme has been the most extensively studied. TXR-1, aspurified from tissues such as placenta, liver, or thymus, and expressedin recombinant form, possesses wide substrate specificity and generallyhigh reactivity with electrophilic agents. The catalytic site of TXR-1encompasses an easily accessible selenocysteine (Sec) residue situatedwithin a C-terminal motif -Gly-Cys-Sec-Gly-COOH. See, e.g., Zhong, L.,et al., Rat and calf thioredoxin reductase are homologous to glutathionereductase with a carboxyl-terminal elongation containing a conservedcatalytically active penultimate selenocysteine residue. J. Biol. Chem.273:8581-8591 (1998). Together with the neighboring cysteine, it forms aredox-active selenenylsulfide/selenolthiol motif that receives electronsfrom a redox-active -Cys-Val-Asn-Val-Gly-Cys-motif present in theN-terminal domain of the other subunit in the dimeric enzyme. See, e.g.,Sandalova, T., et al., Three-dimensional structure of a mammalianthioredoxin reductase: implications for mechanism and evolution of aselenocysteine-dependent enzyme. Proc. Natl. Acad. Sci. USA 98:9533-9538(2001). Substrates of the TXR-1 enzyme, that can be reduced by theselenolthiol motif, include: protein disulfides such as those inthioredoxin; NK-lysin; protein disulfide isomerase; calcium-bindingproteins-1 and -2; and plasma glutathione peroxidase; as well as smallmolecules such as 5,5′-dithiobis(2-nitrobenzoate) (DTNB); alloxan;selenodiglutathione; methylseleninate; S-nitrosoglutathione; ebselen;dehydroascorbate; and alkyl hydroperoxides. See, e.g., Amk, E. S., etal., Preparation and assay of mammalian thioredoxin and thioredoxinreductase. Method. Enzymol. 300:226-239 (1999). Additionally, severalquinone compounds can be reduced by the enzyme and one-electron reducedspecies of the quinones may furthermore derivatize the selenolthiolmotif, thereby inhibiting the enzyme. The highly accessibleselenenylsulfide/selenolthiol motif of the enzyme is extraordinarilyreactive and can be rapidly derivatized by various electrophiliccompounds.

Due to the many important functions of TXR, it is not surprising thatits inhibition could be deleterious to cells due to an inhibition of thewhole thioredoxin system. Moreover, in addition to a general inhibitionof the thioredoxin system as a mechanism for cytotoxicity, it has alsobeen shown that selenium-compromised forms of TXR may directly induceapoptosis in cells by a gain of function. See, e.g., Anestal, K., etal., Rapid induction of cell death by selenium-compromised thioredoxinreductase 1, but not by the fully active enzyme containingselenocysteine. J. Biol. Chem. 278:15966-15672 (2003). The signalingmechanisms of this apoptotic induction have not been presentlyelucidated. It is clear, however, that electrophilic compoundsinhibiting TXR may have significant cellular toxicity as a result ofthese effects. From these findings it may surmised that TXR inhibitionmay be regarded as a potentially important mechanism by which severalalkylating agents and various chemotherapeutic agents (e.g., themonohydrated complex of cisplatin, oxaliplatin, etc.) commonly utilizedin anticancer treatment, may exert their cytotoxic effects.

Thioredoxin (TX)

Thioredoxins (TXs) are proteins that act as antioxidants by facilitatingthe reduction of other proteins by cysteine thiol-disulfide exchange.While glutaredoxins mostly reduce mixed disulfides containingglutathione, thioredoxins are involved in the maintenance of proteinsulfhydryls in their reduced state via disulfide bond reduction. See,e.g., Print, W. A., et al., The role of the thioredoxin and glutaredoxinpathways in reducing protein disulfide bonds in the Escherichia colicytoplasm. J. Biol. Chem. 272:15661-15667 (1996). Thiol-disulfideexchange is a chemical reaction in which a thiolate group (S) attacks asulfur atom of a disulfide bond (—S—S—). The original disulfide bond isbroken, and its other sulfur atom is released as a new thiolate, thuscarrying away the negative charge. Meanwhile, a new disulfide bond formsbetween the attacking thiolate and the original sulfur atom. Thetransition state of the reaction is a linear arrangement of the threesulfur atoms, in which the charge of the attacking thiolate is sharedequally. The protonated thiol form (—SH) is unreactive (i.e., thiolscannot attack disulfide bonds, only thiolates). In accord,thiol-disulfide exchange is inhibited at low pH (typically, <8) wherethe protonated thiol form is favored relative to the deprotonatedthiolate form. The pK_(a) of a typical thiol group is approximately 8.3,although this value can vary as a function of the environment. See,e.g., Gilbert, H. F., Molecular and cellular aspects of thiol-disulfideexchange. Adv. Enzymol. 63:69-172 (1990); Gilbert, H. F.,Thiol/disulfide exchange equilibria and disulfide bond stability. Meth.Enzymol. 251:8-28 (1995).

Thiol-disulfide exchange is the principal reaction by which disulfidebonds are formed and rearranged within a protein. The rearrangement ofdisulfide bonds within a protein generally occurs via intra-proteinthiol-disulfide exchange reactions; a thiolate group of a cysteineresidue attacks one of the protein's own disulfide bonds. This processof disulfide rearrangement (known as disulfide shuffling) does notchange the number of disulfide bonds within a protein, merely theirlocation (i.e., which cysteines are actually bonded). Disulfidereshuffling is generally much faster than oxidation/reduction reactions,which actually change the total number of disulfide bonds within aprotein. The oxidation and reduction of protein disulfide bonds in vitroalso generally occurs via thiol-disulfide exchange reactions. Typically,the thiolate of a redox reagent such as glutathione or dithiothreitol(DTT) attacks the disulfide bond on a protein forming a mixed disulfidebond between the protein and the reagent. This mixed disulfide bond whenattacked by another thiolate from the reagent, leaves the cysteineoxidized. In effect, the disulfide bond is transferred from the proteinto the reagent in two steps, both thiol-disulfide exchange reactions.

Thioredoxin (TX) was originally described in 1964 as a hydrogen donorfor ribonucleotide reductase which is an essential enzyme for DNAsynthesis in Escherichia coli. Human thioredoxin was originally clonedas a cytokine-like factor named adult T cell leukemia (ATL)-derivedfactor (ADF), which was first defined as an IL-2 receptor α-chain(IL-2Ra, CD25)-inducing factor purified from the supernatant of human Tcell leukemia virus type-1 (HTLV-1)-transformed T cell ATL2 cells. See,e.g., Yordi, J., et al., ADF, a growth-promoting factor derived fromadult T cell leukemia and homologous to thioredoxin: possibleinvolvement of dithiol-reduction in the IL-2 receptor induction. EMBO J.8:757-764 (1989).

Proteins sharing the highly conserved -Cys-Xxx-Xxx-Cys- and possessingsimilar three-dimensional structure (i.e., the thioredoxin fold) areclassified as belonging to the thioredoxin family. In the cytosol,members of the thioredoxin family include: the “classical cytosolic”thioredoxin 1 (TX-1) and glutaredoxin 1. In the mitochondria, familymembers include: mitochondrial-specific thyroxin 2 (TX-2) andglutaredoxin 2. Thioredoxin family members in the endoplasmic reticulum(ER) include: protein disulfide isomerase (PDI); calcium-binding protein1 (CaBP1); ERp72; TX-related transmembrane protein (TMX); ERdj5; andsimilar proteins. Macrophage migration inhibitory factor (MIF) is apro-inflammatory cytokine which was originally described as a solublefactor expressed by activated T cells in delayed-type hypersensitivity.See, e.g., Morand, E. F., et al., MIF: a new cytokine link betweenrheumatoid arthritis and atherosclerosis. Nat. Rev. Drug Discov.5:399-411 (2006). MIF also possesses a redox-active catalytic site andexhibits disulfide reductase activity. See, e.g., Kleeman, R., et al.,Disulfide analysis reveals a role for macrophage migration inhibitoryfactor (MIF) as thiol-protein oxidoreductase. J. Mol. Biol. 280:85-102(1998). MIF has pro-inflammatory functions, whereas thioredoxin 1 (TX-1)exhibits both anti-inflammatory and anti-apoptotic functions. TX-1 andMIF control their expression reciprocally, which may explain theiropposite functions. However, TX-1 and MIF also share various similarcharacteristics. For example, both have a similar molecular weight ofapproximately 12 kDa and are secreted by a leaderless export pathway.They both share the same interacting protein such as Jun activationdomain-binding protein 1 (JABI) in cells. Glycosylation inhibitoryfactor (GIF), which was originally reported as a suppressive factor forIgE response, is a posttranslationally-modified MIF with cysteinylationat Cys⁶⁰. The biological difference between MIF and GIF may be explainedby redox-dependent modification, possibly involving TX-1. See, e.g.,Nakamura, H., Thioredoxin and its related molecules: update 2005.Antioxid. Redox Signal. 7:823-828 (2005).

The mammalian thioredoxins (TXs) are a family of 10-12 Kda proteins thatcontain a highly conserved -Trp-Cys-Gly-Pro-Cys-Lys- catalytic site.See, e.g., Nishinaka, Y., et al., Redox control of cellular functions bythioredoxin: A new therapeutic direction in host defense. Arch. Immunol.Ther. Exp. 49:285-292 (2001). The active site sequences is conservedfrom Escherichia coli to humans. Thioredoxins in mammalian cellspossess >90% homology and have approximately 27% overall homology to theE. coli protein.

As previously discussed, the thioredoxins act as oxidoreductases andundergo reversible oxidation/reduction of the two catalytic sitecysteine (Cys) amino acid residues. The most prevalent thioredoxin,TX-1, is involved in a plethora of diverse biological activities. Thereduced dithiol form of TX [TX-(SH)₂] reduces oxidized proteinsubstrates that generally contain a disulfide group; whereas theoxidized disulfide form of TX [TX-(SS)] redox cycles back in anNADPH-dependent process mediated by thioredoxin reductase (TXR), ahomodimer comprised of two identical subunits each having a molecularweight of approximately 55 kDa. The conversion of thioredoxin from thedisulfide form (oxidized) to the dithiol form (reduced) is illustratedin the diagram, below:

Two principal forms of thioredoxin (TX) have been cloned. TX-1 is a105-amino acid protein. In almost all (>99%) of the human form of TX-1,the first methionine (Met) residue is removed by an N-terminus excisionprocess (see, e.g., Giglione, C., et al., Protein N-terminal methionineexcision. Cell. Mol. Life Sci. 61:1455-1474 (2004), and therefore themature protein is comprised of a total of 104 amino acid residues fromthe N-terminal valine (Val) residue. TX-1 is typically localized in thecytoplasm, but it has also been identified in the nucleus of normalendometrial stromal cells, tumor cells, and primary solid tumors.Various types of post-translational modification of TX-1 have beenreported: (i) C-terminal truncated TX-1, comprised of 1-80 or 1-84N-terminal amino acids, is secreted from cells and exhibits morecytokine-like functions than full-length TX-1; (ii) S-Nitrosylation atCys⁶⁹ is important for anti-apoptotic effects; (iii) glutathionylationoccurs at Cys⁷³, which is also the site responsible for the dimerizationinduced by oxidation; (iv) in addition to the original active sitebetween Cys³² Cys³⁵, another dithiol/disuifide exchange is observedbetween and Cys⁶² and Cys⁶⁹, allowing intramolecular disulfideformation; and (v) Cys³⁵ and Cys⁶⁹ are reported to be the target for15-deoxyprostaglandin-J₂. See, e.g., Nakamura, H., Thioredoxin and itsrelated molecules: update 2005. Antioxid. Redox Signal. 7:823-828(2005).

Reduced TX-1, but not its oxidized form or a Cys→Ser catalytic sitemutant, has been shown to bind to various intracellular proteins and mayregulate their biological activities. In addition to NK-κB and Ref-1,TX-1 binds to various isoforms of protein kinase C (PKC); p40 phagocyteoxidase; the nuclear glucocorticoid receptor; and lipocalin. TX-1 alsobinds to apoptosis signal-regulating kinase 1 (ASK 1) in the cytosolunder normal physiological conditions. However, when TX-1 becomesoxidized under oxidative stress, ASK 1 is dissociated from TX-1 and TX-1becomes a homodimer to transduce the apoptotic signal. ASK 1 is anactivator of the JNK and p38 MAP kinase pathways, and is required forTNFα-mediated apoptosis. See, e.g., Saitoh, M., et al., Mammalianthioredoxin is a direct inhibitor of apoptosis signal-regulating kinase1 (ask1). EMBO J. 17:2596-2606 (1998).

Another binding protein for TX-1 is thioredoxin-binding protein 2(TBP-2) which is identical to Vitamin D₃ upregulating protein 1 (VDUP1).TBP-2NDUP1 was originally reported as the product of a gene whoseexpression was upregulated in HL-60 cells stimulated with 1a,25-dihydroxyvitamin D₃. The interaction of TBP-2NDUP1 with TRX wasobserved both in vitro and in vivo. TBP-2/VDUP1 only binds to thereduced form of TRX and acts as an apparent negative regulator of TRX.See, e.g., Nishiyama, A., et al., Identification of thioredoxin-bindingprotein-2/Vitamin D(3) up-regulated protein 1 as a negative regulator ofthioredoxin function and expression. J. Biol. Chem. 274:21645-21650(1999). Although the mechanism is unknown, a reciprocal expressionpattern of TRX and TBP-2 was often reported upon various types ofstimulation. Several highly homologous genes of TBP-2NDUP1 have beenidentified. A TBP-2 homologue, TBP-2-like inducible membrane protein(TLIMP) is a novel VD3 or peroxisome proliferator-activated receptor-γ(PPAR-γ) ligand-inducible membrane-associated protein and plays aregulatory role in cell proliferation and PPAR-γ activation. See, e.g.,Oka, S., et al., Thioredoxin-binding protein 2-like inducible membraneprotein is a novel Vitamin D₃ and peroxisome proliferator-activatedreceptor (PPAR) gamma ligand target protein that regulates PPAR gammasignaling. Endocrinology 147:733-743 (2006). Another TBP-2 homologousgene, DRH1, is reported to be down-regulated in hcpatocellularcarcinoma. See, e.g., Yamamoto, Y., et al., Cloning and characterizationof a novel gene, DRH1, down-regulated in advanced human hepatocellularcarcinoma. Clin. Cancer Res. 7:297-303 (2001). These results indicatethat the familial members of TBP-2 may also play a role in cancersuppression.

TBP-2 also possesses a growth suppressive activity. Overexpression ofTBP-2 was shown to resulted in growth suppression. TBP-2 expression isupregulated by Vitamin D₃ treatment and serum- or IL-2-deprivation, thusleading to growth arrest. TBP-2 is found predominantly in the nucleus.TBP-2 mRNA expression is down-regulated in several tumors (see, e.g.,Butler, L. M., et al., The histone deacetylase inhibitor SAHA arrestscancer cell growth, up-regulates thioredoxin-binding protein-2 anddown-regulates thioredoxin. Proc. Natl. Acad. Sci. USA 99:11700-11705(2002)) and lymphoma (see, e.g., Tome, M. E., et al., A redox signaturescore identifies diffuse large B-cell lymphoma patients with poorprognosis. Blood 106:3594-3601 (2005)), suggesting a close associationbetween the expression reduction and tumorigenesis. TBP-2 expression isalso downregulated in melanoma metastasis. See, e.g., Goldberg, S. F.,et al., Melanoma metastasis suppression by chromosome 6: evidence for apathway regulated by CRSP3 and TXNIP. Cancer Res. 63:432-440 (2003).

Loss of TBP-2 seems to be an important step of human T cell leukemiavirus 1 (HTLV-1) transformation. In an in vitro model, HTLV-1-infectedT-cells required IL-2 to proliferate in the early phase oftransformation, but subsequently lost cell cycle control in the latephase, as indicated by their continuous proliferative state in theabsence of IL-2. The change of cell growth phenotype has been suggestedto be one of the oncogenic transformation processes. See, e.g., Maeda,M., et al., Evidence for the interleukin-2 dependent expansion ofleukemic cells in adult T cell leukemia. Blood 70:1407-1411 (1987). Theexpression of TBP-2 is lost in HTLV-1-positive IL-2-independent T celllines (due to the DNA methylation and histone deacetylation); but ismaintained in HTLV-1-positive IL-2-dependent T cell lines, as well as inHTLV-1-negative T cell lines. See, e.g., Ahsan, M. K., et al., Loss ofinterleukin-2-dependancy in HTLV-1-infected T cells on gene silencing ofthioredoxin-binding protein-2. Oncogene 25:2181-2191 (2005).Additionally, the murine knock-out HcB-19 strain, which has aspontaneous mutation in TBP-2/Txnip/VDUP1 gene, has been reported tohave an increased incidence of hepatocellular carcinoma (HCC), showingthat TBP-2NDUP1 is a potential tumor suppressor gene candidate, in vivo.See, e.g., Sheth, S. S., et al., Thioredoxin-interacting proteindeficiency disrupts the fasting-feeding metabolic transition. J. LipidRes. 46:123-134 (2005). The same HcB-19 mice also exhibited decreased NKcells and reduced tumor rejection. TBP-2 was also found to interact withvarious cellular target such as JAB1 and FAZF, and may be a component ofa transcriptional repressor complex. See, e.g., Lee, K. N., et al.,VDUP1 is required for the development of natural killer cells. Immunity22:195-208 (2005). However, the precise mechanism of its molecularaction remains to be elucidated.

TX-2 is a 166-amino acid protein that contains a 60-amino acid residueN-terminal translocation sequence that directs it to the mitochondria.See, e.g., Spyroung, M., et al., Cloning and expression of a novelmammalian thioredoxin. J. Biol. Chem. 272: 2936-2941 (1997). TX-2 isexpressed uniquely in mitochondria, where it regulates the mitochondrialredox state and plays an important role in cell proliferation.TX-2-deficient cells fall into apoptosis via the mitochondria-mediatedapoptosis signaling pathway. See, e.g., Noon, L., et al., The absence ofmitochondrial thioredoxin-2 causes massive apoptosis and early embryoniclethality in homozygous mice. Mol. Cell. Biol. 23:916-922 (2003). TX-2was found to form a complex with cytochrome c localized in themitochondrial matrix, and the release of cytochrome c from themitochondria was significantly enhanced when expression of TX-2 wasinhibited. The overexpression of TX-2 produced resistance tooxidant-induced apoptosis in human osteosarcoma cells, indicating acritical role for the protein in protection against apoptosis inmitochondria. See, e.g., Chen, Y., et al., Overexpressed humanmitochondrial thioredoxin confers resistance to oxidant-inducedapoptosis in human osteosarcoma cells. J. Biol. Chem. 277:33242-33248(2002).

As both TX-1 and TX-2 are known regulators of the manifestation ofapoptosis under redox-sensitive capases, their actions may becoordinated. However, the functions of TX-1 and TX-2 do not seem to becapable of compensating for each other completely, since TX-2 knockoutmice were found be embryonically lethal. See, e.g., Noon, L., et al.,The absence of mitochondrial thioredoxin-2 causes massive apoptosis andearly embryonic lethality in homozygous mice. Mol. Cell. Biol.23:916-922 (2003). Moreover, the different subcellular locations of boththe thioredoxin reductase (TXR) and thioredoxin (TX) subtypes suggestthat the cytoplasmic and mitochondrial systems may play different roleswithin cells. See, e.g., Powis, G. and Monofort, W. R. Properties andbiological activities of thioredoxins. Ann. Rev. Pharmacol. Toxicol.41:261-295 (2001).

IV. Biological Activities of the TRX/TX System Physiological and EffectsModulated by Thioredoxin (TX) and Related Proteins

Mammalian cells contain a glutathione (GSH)/glutaredoxin system and athioredoxin (TX)/thioredoxin reductase (TXR) system as the two majorantioxidant systems. The intracellular concentration of GSH isapproximately 1-10 milliMolar (mM) in mammalian cells, whereas thenormal reported intracellular concentration of TX is approximately 0.1-2μM. Accordingly, TX may initially appear as a minor component as anintracellular antioxidant. However, TX is a major enzyme supplyingelectrons to peroxiredoxins or methionine sulfoxide reductases, and actsas general protein disulfide reductase. TX knock-out mice are embryoniclethal (see, e.g., Matsui, M., et al., Early embryonic lethality causedby targeted disruption of the mouse thioredoxin gene. Dev. Biol.178:179-185 (1996)), thus illustrating that the TX/TXR system is playingan essential survival role in mammalian cells. This importance may beexplained by TX playing a crucial role in the interaction with specifictarget proteins including, but not limited to, the inhibition ofapoptosis signal regulation kinase I (ASK1) activation (see, e.g.,Saitoh, M., et al., Mammalian thioredoxin is a direct inhibitor ofapoptosis signal-regulation kinase 1 (ASK1). EMBO J. 17:2596-2606(1998)) and in the regulation of DNA binding activity of transcriptionalfactors such as AP-1, NF-κB and p53 for the transcriptional control ofessential genes (see, e.g., Nakamura, H., et al., Redox regulation ofcellular activation. Ann. Rev. Immunol. 15:351-369 (1997)). For example,during oxidative stress TX-1 translocates from the cytosol into thenucleus where it augments DNA-binding activity of these aforementionedtranscriptional factors. Alternately, the role of TX in the defenseagainst cellular oxidative stress or to supply the “building blocks” forDNA synthesis, via ribonucleotide reductase, is equally essential. TX-1and the 14 Kda TX-like protein (TRP14) reactivates PTEN (a proteintyrosine phosphatase which reverses the action ofphosphoinositide-3-kinase) by the reduction of the disulfide which isreversibly induced by hydrogen peroxide. See, e.g., Jeong, W., et al.,Identification and characterization of TRP14, a thioredoxin-relatedprotein of 14 Kda. J. Biol. Chem. 279:3142-3150 (2004). Exogenous TX-1has been shown to be capable of entering cells and attenuateintracellular reactive oxygen species (ROS) generation and cellularapoptosis. See, e.g., Kondo, N, et al., Redox-sensing release of humanthioredoxin from T lymphocytes with negative feedback loops. J. Immunol.172:442-448 (2004). Additionally, HMG-CoA reductase inhibitors (commonlyutilized for the prevention of atherosclerosis) have also been shown toaugment S-Nitrosylation of TX-1 at Cys⁶⁹ and reduce oxidative stress.See, e.g., Haendeler, J., et al., Antioxidant effects of statins viaS-nitrosylation and activation of thioredoxin in endothelial cells.Circulation 110:856-861 (2004).

The TX/TXR System as a Cofactor in DNA Synthesis

The TX/TXR-coupled system plays a critical role in the generation ofdeoxyribonucleotides which are needed in DNA synthesis and essential forcell proliferation. TX provides the electrons needed in the reduction ofribose by ribonucleotide reductase, an enzyme that catalyzes theconversion of nucleotide diphosphates into deoxyribonucleotides.Ribonucleotide reductase is necessary for DNA synthesis and cellproliferation. Diaziquone and doxorubicin have been shown to inhibit theTR/TXR system resulting in a concentration-dependent inhibition ofcellular ribonucleotide reductase activity in human cancer cells. See,e.g., Mau, B., et al., Inhibition of cellular thioredoxin reductase bydiaziquone and doxorubicin. Biochem. Pharmacol. 43:1621-1626 (1992).Similarly, the glutaredoxin/glutathione-coupled reaction also providesreducing equivalents for ribonucleotide reductase. For example,depletion of glutathione has been shown to inhibit DNA synthesis andinduce apoptosis in a number of cancer cell lines. See, e.g.,Dethlefsen, L. A., et al., Toxic effects of acute glutathione depletionby on murine mammary carcinoma cells. Radiat. Res. 114:215-224 (1988).

The role of the TX/TXR System in Cellular Apoptosis

TX-1 was shown to prevent apoptosis (programmed cell death) when addedto the culture medium of lymphoid cells or when its gene is transfectedinto these cells. Murine WEH17.2 lymphoid cells underwent apoptosis whenexposed to the glucocorticoid dexamethasone or the topoisomerase Iinhibitor etoposide and, to a lesser extent, when exposed to the kinaseinhibitor staurosporine or thapsigarin, an inhibitor of intracellularcalcium uptake. See, e.g., Powis, G., et al., Thioredoxin control ofcell growth and death and the effects of inhibitors. Chem. Biol.Interact. 111:23-34 (1998). TX levels in the cytoplasm and nucleus wereincreased following stable transfection of these cells with human TX-1,and as a result the transfected cells showed resistance to apoptosiswhen exposed to dexamethasone and the other cytotoxic agents. Thepattern of apoptosis inhibition with TX-1 transfection was similar tothat following transfection with the bcl-2 anti-apoptotic oncogene. Incooperation with redox factor-1, TX-1 induces p53-dependent p-21transactivation leading to cell-cycle arrest and DNA repair. See, e.g.,Ueda, S., et al., Redox control of cell death. Antioxid. Redox Signal.4:405-414 (2002). In addition, TX-1 regulates the signaling forapoptosis by suppressing the activation of apoptosis signal-regulationkinase-1 (ASK-1). See, e.g., Nakamura, H., et al., Redox regulation ofcellular activation. Ann. Rev. Immunol. 15:351-369 (1997).

The specific mechanism(s) by which TX-2 imparts resistance tochemotherapy apoptosis in cancer cells has not been fully elucidated.Based on the current studies, one may postulate, however, that itappears increases in cellular reductive power allows ongoing protectiveand/or reparative reduction of proteins, DNA, cell membranes orcarbohydrates that have been damaged or would otherwise be damaged byoxidative chemical species, thus counteracting of the induced cellularapoptosis from the chemotherapy and/or radiation therapy. The analogousglutaredoxin/glutathione system may also prevent apoptosis. In eitherinstance, there is a lack of apoptotic sensitivity to normal treatmentinterventions that appears to be mediated by the increased TX-2 and byglutaredoxin pathways. In the glutaredoxin mediated pathway, as anexample, glutathione depletion with L-buthionine sulfoximine was shownto inhibit the growth of several breast and prostate cancer cell lines,and in rat R3230Ac mammary carcinoma cells, it markedly increasedapoptosis. It is thought that mitochondrial swelling following depletionof glutathione may be the stimulus for apoptosis in these cells. See,e.g., Bigalow, J. E., et al., Glutathione depletion or radiationtreatment alters respiration and induces apoptosis in R3230Ac mammarycarcinoma. Adv. Exp. Med. Biol. 530:153-164 (2003). TX-2 has been shownto be a critical regulator of mitochondrial cytochrome c release andapoptosis. See, e.g., Tanaka, M., et al., Thioredoxin-2 (TX-2) is anessential gene in regulating mitochondrial-dependent apoptosis. EMBO J.21:1695-1701 (2002).

The Role of TX in Stimulating Angiogenesis

Angiogenesis by cancer cells provides a growth and survival advantagethat is localized to the primary as well as secondary (metastatictumors). Malignant tumors are generally poorly vascular, however, withoverexpression of angiogenesis factors, the tumor cells gain betternutrition and oxygenation, thereby promoting proliferation of cancercells and growth of the tumor. Transfection of several different celllines, including human breast cancer MCF-7, human colon cancer HT29, andmurineWEHI7.2 lymphoma cells, with human TX-1 produced significantincreases in secretion of vascular endothelial growth factor (VEGF).See, e.g., Welch, S. J., et al., The redox protein thioredoxin-1increases hypoxia-inducible factor 1α protein expression: TXR-1overexpression results in increased vascular endothelial growth factorproduction and enhanced tumor angiogenesis. Cancer Res. 62:5089-5095(2003). VEGF secretion was increased by 41%-77% under normoxic (20%oxygen) conditions and by 46%-79% under hypoxic (1% oxygen) conditions.In contrast, transfection with a redox-inactive TX mutant (Cys→Ser)partially inhibited VEGF production. When TX-1-transfected WEH17.2 cellswere grown in SCID mice, VEGF levels were markedly increased and tumorangiogenesis (as measured by microvessel vascular density) was alsoincreased by 2.5-fold, relative to wild-type WEH17.2 tumors. Id.Accordingly, there is evidence that the thioredoxin system can increaseVEGF levels in cancer cells.

Role of TX in Stimulating Cell Proliferation

Exposure to TX-1 was shown to stimulate the growth of lymphocytes,fibroblasts, and a variety of leukemic and solid tumor cell lines. See,e.g., Powis, G. and Monofort, W. R. Properties and biological activitiesof thioredoxins. Ann. Rev. Pharmacol. Toxicol. 41:261-295 (2001). Incontrast, the previously discussed Cys→Ser redox mutant at 50-foldhigher concentrations, did not stimulate cell growth. While themechanisms for this proliferative effect are not fully elucidated, thereis evidence that such TX-mediated increases in cell proliferation aremultifactorial, and are related to both the increased production ofvarious cytokines (e.g., IL-1, IL-2, and tumor necrosis factor α (TNFα))and the potentiation of growth factor activity (e.g., basic fibroblastgrowth factor (bFGF)). Additionally, there is thought to also beincreased DNA synthesis and transcription, as well.

The Antioxidant Effects of TX

Glutathione peroxidase and membrane peroxidases play a highly importantrole in protecting cells against the damaging effects of reactive oxygenspecies (ROS) including, but not limited to, oxygen radicals andperoxides. See, e.g., Bigalow, J. E., et al., The importance of peroxideand superoxide in the x-ray response. Int. J. Radiat. Oncol. Biol. Phys.22:665-669 (1992). These enzymes utilize use thiol groups as an electronsource for scavenging reactive oxygen species (ROS), and in the process,form homo- or heterodimers with other peroxidases through the formationof disulfide bonds with conserved cysteine residues. TX producesantioxidant effects primarily by serving as an electron donor forthioredoxin peroxidases. Accordingly, by the reduction of oxidizedperoxidases, TX restores the enzyme to its monomeric form, which allowsthe enzyme to continue its oxyradical scavenging.

TX may also increase the expression of thioredoxin peroxidase. Forexample, in MCF-7 human breast cancer cells stably transfected withTX-1, mRNA for thioredoxin peroxidase was doubled relative to wild-typeand empty-vector transformed cells, and Western blots showed increasedprotein levels as well. Moreover, TX-1 transfected murine WEH17.2 cellswere more resistant to peroxide-induced apoptosis than wild-type andempty-vector transformed cells. However, TX-1 transfection did notprotect the cells from apoptosis induced by dexamethasone orchemotherapeutic agents. See, e.g., Berggren, M. I., et al., Thioredoxinperoxidase-1 is increase in thioredoxin-1 transfected cells and resultsin enhanced protection against apoptosis caused by hydrogen peroxide,but not by other agents including dexamethasone, etoposide, anddeoxorubin. Arch. Biochem. Biophys. 392:103-109 (2001).

The Role of TX in Stimulating Transcription Factor Activity

Thioredoxin (TX) increases the DNA-binding activity of a number oftranscription factors (e.g., NF-κB, AP-1, and AP-2) and nuclearreceptors (e.g., glucocorticoid and estrogen receptors). See, e.g.,Nishinaka, Y., et al., Redox control of cellular functions bythioredoxin: A new therapeutic direction in host defense. Arch. Immunol.Ther. Exp. 49:285-292 (2001). By way of non-limiting example, withregard to NF-κB, TX reduces the Cys residue of the p50 subunit in thenucleus, thus allowing it to bind to DNA. See, e.g., Mau, B., et al.,Inhibition of cellular thioredoxin reductase by diaziquone anddoxorubicin. Biochem. Pharmacol. 43:1621-1626 (1992). In the cytoplasm,however, TX paradoxically interferes with NF-κB by blocking dissociationof the endogenous inhibitor IκB and interfering with signaling to IκBkinases. See, e.g., Hirota, K., et al., Distinct roles of thioredoxin inthe cytoplasm and in the nucleus: A two-step mechanism of redoxregulation of transcription factor nf-κB. J. Biol. Chem. 274:27891-27897(1999). The effect of TX on some transcription factors is mediated viareduction of Ref-1, a 37 kDa protein that also possesses DNA-repairendonuclease activity. For example, TX reduces Ref-1, which in turnreduces cysteine residues within the fos and jun subunits of AP-1 topromote DNA binding. The redox activity of Ref-1 is found in itsN-terminal domain, whereas its DNA repair activity is located amongC-terminal sequences.

TX Binding to Cellular Proteins

Reduced TX-1, but not its oxidized form or a catalytic site Cys→Serredox inactive mutant, binds to a variety of cellular proteins and mayregulate their biological activities. See, e.g., Powis, G. and Monofort,W. R. Properties and biological activities of thioredoxins. Ann. Rev.Pharmacol. Toxicol. 41:261-295 (2001). In addition, to NK-κB and Ref-1,TX binds to: (i) apoptosis signal-regulating kinase 1 (ASK1), (ii)various isoforms of protein kinase C (PKC), (iii) p40 phagocyte oxidase,(iv) the nuclear glucocorticoid receptor, and (v) lipocalin. ASK1, forexample, is an activator of the JNK and p38 MAP kinase pathways and isrequired for TFNα-mediated apoptosis. See, e.g., Ichijo, H., et al.,Induction of apoptosis by ask1, a mammalian map kinase that activatesjnk and p38 signaling pathways. Science 275:90-94 (1997). TX binds to asite at the N-terminal of ASK1, thus inhibiting the kinase activity andblocking ASK1-mediated apoptosis. See, e.g., Saitoh, M., et al.,Mammalian thioredoxin is a direct inhibitor of apoptosissignal-regulation kinase 1 (ask1). EMBO J. 17:2596-2606 (1998). Underconditions of oxidative stress, however, reactive oxygen species areproduced that oxidize the TX, thus promoting its dissociation from ASK1and leading to the concomitant activation of ASK1.

TX/TXR Expression in Cancer

Various extracellular roles of thioredoxin (TX) have been examined incancer. As previously described, TX was originally cloned as acytokine-like factor named ADF.

Independently, TX was also identified as an autocrine growth factornamed 3B6-IL1 produced by Epstein-Barr virus-transformed B cells (see,e.g., Wakasugi, H., et al., Epstein-Barr virus-containing B-cell lineproduces an interleukin 1 that it uses as a growth factor. Proc. Natl.Acad. Sci. USA 84:804-808 (1987)) or as a B cell growth factor namedMP6-BCGF produced by the T cell hybridoma MP6 (see, e.g., Rosen A, etal., A CD4+ T cell line-secreted factor, growth promoting for normal andleukemic B cells, identified as thioredoxin. Int. Immunol. 7:625-33(1995)). Moreover, eosinophil cytotoxicity-enhancing factor (ECEF) wasfound as a truncated form of TX comprising which is the N-terminal 1-80(or 1-84) residues of TX (Trx80) (see, e.g., Silberstein, D. S., et al.,Human eosinophil cytotoxicity-enhancing factor. Eosinophil-stimulatingand dithiol reductase activities of biosynthetic (recombinant) specieswith COOH-terminal deletions. J. Biol. Chem. 268:913-942 (1993)) and acomponent of “early pregnancy factor” which was an immunosuppressivefactor in pregnant female serum was also identified as TX (see, e.g.,Clarke, F. M., et al., Identification of molecules involved in the“early pregnancy factor” phenomenon. J. Reprod. Fertil. 93:525-539(1991)). These historical reports, collectively, illustrate that TX hasvarious important extracellular functions.

Thioredoxin (TX) expression is increased in a variety of humanmalignancies including, but not limited to, lung cancer, colorectalcancer, cervical cancer, hepatic cancer, pancreatic cancer, andadenocarcinoma. In addition, TX expression has also been associated withaggressive tumor growth. This increase in expression level is likelyrelated to changes in TX protein structure and function. For example, inpancreatic ductal carcinoma tissue, TX levels were found to be elevatedin 24 of 32 cases, as compared to normal pancreatic tissue. Glutaredoxinlevels were increased in 29 of the cases. See, e.g., Nakamura, H., etal., Expression of thioredoxin and glutaredoxin, redox-regulatingproteins, in pancreatic cancer. Cancer Detect. Prey. 24:53-60 (2000).Similarly, tissue samples of primary colorectal cancer or lymph nodemetastases had significantly higher TX-1 levels than normal colonicmucosa or colorectal adenomatous polyps. See, e.g., Raffel, J., et al.,Increased expression of thioredoxin-1 in human colorectal cancer isassociated with decreased patient survival. J. Lab. Clin. Med. 142:46-51(2003).

In two recent studies, TX expression was associated with aggressivetumor growth and poorer prognosis. In a study of 102 primary non-smallcell lung carcinomas, tumor cell TX expression was measured byimmunohistochemistry of formalin-fixed, paraffin-embedded tissuespecimens. See, e.g., Kakolyris, S., et al., Thioredoxin expression isassociated with lymph node status and prognosis in early operablenon-small cell lung cancer. Clin. Cancer Res. 7:3087-3091 (2001). Theabsence of TX expression was significantly associated with lymphnode-negative status (P=0.004) and better outcomes (P<0.05) and wasfound to be independent of tumor stage, grade, or histology. Theinvestigators also concluded that these results were consistent with theproposed role of TX as a growth promoter in some human cancers, andoverexpression may be indicative of a more aggressive tumor phenotype(hence the association of TX overexpression with nodal positivity andpoorer outcomes). In another study of 37 patients with colorectalcancer, TX-1 expression tended to increase with higher Dukes stage(P=0.077) and was significantly correlated with reduced survival(P=0.004). After adjusting for Dukes stage, TX-1 levels remained asignificant prognostic factor associated with survival (P=0.012). See,e.g., Raffel, J., et al., Increased expression of thioredoxin-1 in humancolorectal cancer is associated with decreased patient survival. J. Lab.Clin. Med. 142:46-51 (2003). It should be noted that GSH levels were notdetermined in either of the aforementioned studies.

The relationship between TXR activity and tumor growth is less clear.Tumor cells may not need to increase expression of the TXR enzyme,although its catalytic activity may be increased functionally. Forexample, human colorectal tumors were found to have 2-times higher TXRactivity than normal colonic mucosa. See, e.g., Mustacich, D. and Powis,G., Thioredoxin reductase. Biochem. J. 346:1-8 (2000). TXR has also beenreported to be elevated in human primary melanoma and to show acorrelation with invasiveness. See, e.g., Schallreuter, K. U., et al.,Thioredoxin reductase levels are elevated in human primary melanomacells. Int. J. Cancer 48:15-19 (1991). Further evaluations relating TXRenzyme levels and catalytic activity with cancer stage and outcome arerequired needed to fully elucidate this relationship.

The Role of TX in Stimulating Hypoxia-Inducible Factor (HIF)

Cancer cells are able to adapt to the hypoxic conditions found in nearlyall solid tumors. Hypoxia leads to activation of hypoxia-induciblefactor 1 (HIF-I), which is a transcription factor involved indevelopment of the cancer phenotype. Specifically, HIF binds to hypoxiaresponse elements (HRE) and induces expression of a variety of genesthat serve to promote: (i) angiogenesis VEGF); (ii) metabolic adaptation(e.g., GLUT transporters, hexokinase, and other glycolytic enzymes); and(iii) cell proliferation and survival. HIF is comprised of twosubunits—HIF-1α (that is induced by hypoxia) and HIF-1β (that isexpressed constitutively). TX overexpression has been shown tosignificantly increase HIF-1α under both normoxic and hypoxicconditions, and this was associated with increased HRE activitydemonstrated in a luciferase reporter assay as well as increasedexpression of HRE-regulated genes. HIF may provide tumor cells with asurvival advantage under hypoxic conditions by inducing hexokinase andthus allowing glycolysis to serve as the predominant energy source. Forexample, surgical specimens from patients with metastatic liver cancerhad fewer tumor blood vessels and higher hexokinase expression thanspecimens from hepatocellular carcinoma patients. Hexokinase expressionwas correlated with HIF-1α expression in both populations, and theyco-localized in tumor cells found near necrotic regions.

The TX/TXR System in Cancer Drug Resistance

As previously discussed, mammalian thioredoxin reductase (TXR) isinvolved in a number of important cellular processes including, but notlimited to: cell proliferation, antioxidant defense, and redoxsignaling. Together with glutathione reductase (GR), it is also the mainenzyme providing reducing equivalents to many cellular processes. GR andTXR are flavoproteins of the same enzyme family, but only the latter isa selenoprotein. With the catalytic site containing selenocysteine, TXRmay catalyze reduction of a wide range of substrates, but it can also beeasily targeted by electrophilic compounds due to the extraordinarilyhigh reactivity of the selenocysteine moiety. In a recent studies, theinhibition of TXR and GR by anti-cancer alkylating agents andplatinum-containing compounds was compared to the inhibition of GR. See,e.g., Wang, X., et al., Thioredoxin reductase inactivation as a pivotalmechanism of ifosfamide in cancer therapy. Eur. J. Pharmacol. 579:66-75(2008); Wang, X., et al., Cyclophosphamide as a potent inhibitor oftumor thioredoxin reductase in vivo. Toxicol. Appl. Pharmacol. 218:88-95(2007); Witte, A-B., et al., Inhibition of thioredoxin reductase but notof glutathione reductase by the major classes of alkylating andplatinum-containing anticancer compounds. Free Rad. Biol. Med.39:696-703 (2005). These studies found that: (i) the nitrosourea,carmustine, can inhibit both GR and TXR; (ii) the nitrogen mustards(cyclophosphamide, chlorambucil, and melphalan) and the alkyl sulfonate(busulfan) irreversibly inhibited TXR in a concentration- andtime-dependent manner, but not GR; (iii) the oxazaphosphorine,ifosfamide, inhibited TXR; (iv) the anthracyclines (daunorubicin anddoxorubicin) were not inhibitors of TXR; (v) cisplatin, its monohydratedcomplex, oxaliplatin, and transplatin irreversibly inhibited TXR, butnot GR; and (vi) carboplatin could not inhibit either TXR or GR. Otherstudies have shown that the irreversible inhibition of TXR by quinones,nitrosoureas, and 13-cis-retinoic acid is markedly similar to theinhibition of TXR by cisplatin, oxaliplatin, and transplatin. See, e.g.,Amer, E. S. J., et al., Analysis of the inhibition of mammalianthioredoxin, thioredoxin reductase, and glutaredoxin bycis-diamminedichloroplatinum (II) and its major metabolite, theglutathione-platinum complex. Free Rad. Biol. Med. 31:1170-1178 (2001).

Studies have also shown that the highly accessibleselenenylsulfide/selenolthiol motif of the TXR enzyme can be rapidlyderivatized by a number of electrophilic compounds. See, e.g., Beeker,K, et al., Thioredoxin reductase as a pathophysiological factor and drugtarget. Eur. J. Biochem. 262:6118-6125 (2000). These compounds include,but are not limited to: (i) cisplatin and its glutathione adduct (see,e.g., Amer, E. S. J., et al., Analysis of the inhibition of mammalianthioredoxin, thioredoxin reductase; glutaredoxin bycis-diamminedichlamplatinum (II) and its major metabolite, theglutathioneplatinum complex. Free Rad. Biol. Med. 31:1170-1178 (2001));(ii) dinitrohalobenzenes (see, e.g., Nordberg, J., et al., Mammalianthioredoxin reductase is irreversibly inhibited by dinitrohalobenzenesby alkylation of both the redox active selenocysteine and itsneighboring cysteine residue. J. Biol. Chem. 273:10835-10842 (1998));(iii) gold compounds (see, e.g., Gromer, S., et al., Human placentathioredoxin reductase: Isolation of the selenoenzyme, steady statekinetics, inhibition by therapeutic gold compounds. J. Biol. Chem.273:20096-20101 (1998)); (iv) organochalogenides (see, e.g., Engman, L.,et al., Water-soluble organatellurium compounds inhibit thioredoxinreductase and the growth of human cancer cells. Anticancer Drug. Des.15:323-330 (2000)); (v) different naphthazarin derivatives (see, e.g.,Dessolin, I., et al., Bromination studies of the2.3-dimethylnaphthazarin core allowing easy access to naphthazarinderivatives. J. Org. Chem. 66:5616-5619 (2001)); (vi) certainnitrosoureas (see, e.g., Sehallreuter, K. U., et al., The mechanism ofaction of the nitrosourea anti-tumor drugs and thioredoxin reductase,glutathione reductase and ribonucleotide reductase. Biochim. Biophys.Acta 1054:14-20 (1990)); and (vii) general thiol or selenol alkylatingagents such as C-vinylpyridine, iodoacetamide or iodoacetic acid (see,e.g., Nordberg, J., et al., Mammalian thioredoxin reductase isirreversibly inhibited by dinitrohalobenzenes by alkylation of both theredox active selenocysteine and its neighboring cysteine residue. J.Biol. Chem. 273:10835-10842 (1998)).

Similarly, several lines of evidence suggest that thioredoxin (TX) mayalso be necessary, but is not sufficient in toto, for conferringresistance to many chemotherapeutic drugs. This evidence includes, butis not limited to: (i) the resistance of adult T-cell leukemia celllines to doxorubicin and ovarian cancer cell lines to cisplatin has beenassociated with increased intracellular TX-1 levels; (ii) hepatocellularcarcinoma cells with increased TX-1 levels were less sensitive cisplatin(but not less sensitive to doxorubicin or mitomycin C); (iii) TX-1 mRNAand protein levels were increased by 4- to 6-fold in bladder andprostate cancer cells made resistant to cisplatin, but lowering TX-1levels with an antisense plasmid restored sensitivity to cisplatin andincreased sensitivity to several other cytotoxic drugs; (iv) TX-1 levelswere elevated in cisplatin-resistant gastric and colon cancer cells; and(v) stable transfection of fibrosarcoma cells with TX-1 resulted inincreased cisplatin resistance. See, e.g., Biaglow, J. E. and Miller, R.A., The thioredoxin reductase/thioredoxin system. Cancer Ther. 4:6-13(2005).

Glutathione may also play a role in anti-cancer drug resistance.Glutathione-S-transferases catalyze the conjugation of glutathione tomany electrophilic compounds, and can be upregulated by a variety ofcancer drugs. Glutathione-S-transferases possess selenium-independentperoxidase activity. Mg also has glutaredoxin activity. Some agents aresubstrates for glutathione-S-transferase and are directly inactivated byglutathione conjugation, thus leading to resistance. Examples of enzymesubstrates include melphalan, carmustine (BCNU), and nitrogen mustard.In a panel of cancer cell lines, glutathione-S-transferase expressionwas correlated inversely with sensitivity to alkylating agents. Otherdrugs that upregulate glutathione-S-transferase may become resistant,because the enzyme also inhibits the MAP kinase pathway. These agentsrequire a functional MAP kinase, specifically JNK and p38 activity, toinduce an apoptotic response. See, e.g., Townsend, D. M. and Tew, K. D.,The role of glutathione-S-transferase in anti-cancer drug resistance.Oncogene 22:7369-7375 (2003).

Targeting TX/TXR-Coupled Reactions

The biological activities of TX/TRX and their apparent relevance toaggressive tumor growth suggest that this system may be an attractivetarget for cancer therapy. Either individual enzymes or substrates canbe altered. In cells that do not contain glutaredoxin, depletion ofhexose monophosphate shunt (HMPS)-generated NADPH or, alternately,direct interaction with TX or TRX may prove to be viable approaches toblocking HMPS/TX/TRX-coupled reactions. In cells where glutaredoxin ispresent, its reducing activity also may need to be targeted throughdepletion of glutathione.

Thioredoxin in Plasma or Serum as an Oxidative Metabolism BiologicalMarker

Thioredoxin 1 (TX) is released by cells in response to changes inoxidative metabolism. See, e.g., Kondo N, et al., Redox-sensing releaseof human thioredoxin from T lymphocytes with negative feedback loops. J.Immunol. 172:442-448 (2004). Plasma or serum levels of TX are measurableby a sensitive sandwich enzyme-linked immunosorbent assay (ELISA). Serumplasma levels of TX are good markers for changes in oxidative metabolismin a variety of disorders. See, e.g., Burke-Gaffney, A., et al.,Thioredoxin: friend or foe in human diseases? Trends Pharmacol. Sci.26:398-404 (2004). For example, plasma levels of TRX are elevated inpatients with acquired immunodeficiency syndrome (AIDS) and negativelycorrelated with the intracellular levels of GSH, suggesting that theHIV-infected individuals with AIDS. See, e.g., Nakamura, H., e t al.,Elevation of plasma thioredoxin levels in HIV-infected individuals. Int.Immunol. 8:603-611 (1996). In patients with type C chronic hepatitis,serum levels of TRX and ferritin are good markers for the efficacy ofinterferon therapy. See, e.g., Sumida, Y., et al., Serum thioredoxinlevels as an indicator of oxidative stress in patients with hepatitis Cvirus infection. J. Hepatol. 33:616-622 (2001). In the case of cancer,serum levels of TRX are elevated in patients with hepatocellularcarcinoma (see, e.g., Miyazaki, K., et al., Elevated serum levels ofserum thioredoxin in patients with hepatocellular carcinoma. Biotherapy11:277-288 (1998)) and pancreatic cancer (see, e.g., Nakmura, H., etal., Expression of thioredoxin and glutaredoxin, redox-regulatingproteins, in pancreatic cancer. Cancer Detect. Prev. 24:53-40 (2000)).The serum levels of TX decrease after the removal of the main tumor,suggesting that cancer tissues are the main source of the elevated TX inserum. See, e.g., Miyazaki, K., et al., Elevated serum levels of serumthioredoxin in patients with hepatocellular carcinoma. Biotherapy11:277-288 (1998).

The Use of TX Therapy in Cancer Patients

Since TX shows anti-inflammatory effect in circulation, the clinicalapplication of TX therapy is now planned, especially because TX has beenshown to block neutrophil infiltration into the inflammatory site. Forexample, the administration of recombinant human TX (rhTX) inhibitsbleomycin or inflammatory cytokine-induced interstitial pneumonia. See,e.g., Hoshino, T., et al., Redox-active protein thioredoxin preventsproinflammatory cytokine- or bleomycin-induced lung injury. Am. J.Respir. Crit. Care Med. 168:1075-1083 (2003). Therefore, acuterespiratory distress syndrome (ARDS)/acute lung injury (ALI) is onedisorder which is a good target for TX therapy. ARDS/ALI is caused byvarious etiologies including anti-cancer agents such as gefitinib, amolecular-targeted agent that inhibits epidermal growth factor receptor(EGFR) tyrosine kinase. The safety of TX therapy in cancer patients incurrently being examined. Although the intracellular expression of TX incancer tissues is associated with, e.g., resistance to anti-canceragents (see, e.g., Yokomizo, A., et al., Cellular levels of thioredoxinassociated with drug sensitivity to cisplatin, mitomycin C, deoxrubicin,and etoposide. Cancer Res. 55:4293-4296 (1995); Sasada, T., et al.,Redox control and resistance to cis-diamminedichloroplatinum (II)(CDDP); protective effect of human thioredoxin against CDDP-inducedcytotoxicity. J. Clin. Investig. 97:2268-2276 (1996)), there is noevidence showing that exogenously administered rhTRX promotes the growthof cancer. For example, there is no promoting effect of administeredrhTRX on the growth of the tumor planted in nude mice. In addition,administered rhTRX has no inhibitory effect on the anti-cancer agent tosuppress the tumor growth in nude mice. It may be explained by that thecellular uptake of exogenous TRX is quite limited and administered TRXin plasma immediately becomes the oxidized form which has no tumorgrowth stimulatory activity as previously mentioned.

Thioredoxin 1 (TX) expression is enhanced in cancer tissues and nowinhibitors for TX and/or thioredoxin reductase (TXR) are studied as newanti-cancer agents. See, e.g., Powis, G., Properties and biologicalactivities of thioredoxin. Annu. Rev. Phamacol. Toxicol. 41:261-295(2001). From this aspect, TX gene therapy may be dangerous incancer-bearing patients. In contrast, the administration of rhTX may besafe and applicable even in cancer-bearing patients to attenuate theinflammatory disorders associated with the leukocyte infiltration.

It should also be noted that, the Japan Phase III non-small cell lungcarcinoma (NSCLC) Clinical Trial and the United States (U.S.) Phase IINSCLC Clinical Trial, that are discussed and described in the presentinvention represent controlled clinical evidence of a survival increasecaused a thioredoxin and/or glutaredoxin inactivating or modulatingmedicament (that act pharmacologically in the manner of the oxidativemetabolism-affecting Formula (I) compounds of the present invention).These two aforementioned clinical trials will be fully discussed in alater section. However, it is observed from the data from both of thesecontrolled clinical trials that there is a marked increase in patientsurvival, especially in the non-small cell lung carcinoma,adenocarcinoma sub-type patients receiving a Formula (I) compound of thepresent invention. For example, there was an increase in median survivaltime of approximately 138 days (i.e., 4.5 months) and approximately 198days (i.e., 6.5 months) for adenocarcinoma patients in the Tavocept armof the Japan Phase III NSCLC Clinical Trial and the U.S. Phase II NSCLCClinical Trial, respectively.

Various representative Formula (I) compounds of the present inventionhave been synthesized and purified. Additionally, disodium2,2′-dithio-bis ethane sulfonate (also referred to in the literature asTavocept™, dimesna, and BNP7787), a Formula (I) compound of the presentinvention, has been introduced into Phase I, Phase II, and Phase IIIclinical testing in patients, as well as in non-clinical testing, by theAssignee, BioNumerik Pharmaceuticals, Inc., with guidance provided bythe Applicant of the instant invention. In addition, this compound hasbeen utilized in a multicenter, randomized, Phase II clinical trialinvolving patients with advanced Stage IIIB/IV non-small cell lungcarcinoma (NSCLC), including adenocarcinoma (the U.S. Phase II NSCLCClinical Trial). Data from the aforementioned recent Phase II and PhaseIII clinical trials utilizing disodium 2,2′-dithio-bis ethane sulfonate(Tavocept™) with chemotherapeutic agent(s) have demonstrated the abilityof disodium 2,2′-dithio-bis ethane sulfonate to markedly increase thesurvival time of individuals with non-small cell lung carcinoma (NSCLC),including the adenocarcinoma sub-type. In brief, experimental evidencesupports the finding that disodium 2,2′-dithio-bis ethane sulfonatefunctions to increase patient survival time by increasing oxidativemetabolism within tumor cells in a selective manner.

The Applicant of the present invention has previously disclosed the useof disodium 2,2′-dithio-bis ethane sulfonate and other dithioethers to:(i) mitigate nephrotoxicity (see, e.g., U.S. Pat. Nos. 5,789,000;5,866,169; 5,866,615; 5,866,617; and 5,902,610) and (ii) mitigateneurotoxicity (see, e.g., Published U.S. Patent Application No.2003/0133994); all of which are incorporated herein by reference intheir entirety. However, as previously stated, the novel approach of thepresent invention involve compositions, methods, and kits which cause anincrease in survival time of cancer patients, wherein the cancer either:(i) overexpresses thioredoxin and/or glutaredoxin and/or (ii) exhibitsevidence of thioredoxin- or glutaredoxin-mediated resistance to one ormore chemotherapeutic agents.

The present invention discloses and claims: (i) compositions which causean increase in the time of survival in patients with cancer; wherein thecancer either overexpresses thioredoxin or glutaredoxin or exhibits orpossesses thioredoxin- or glutaredoxin-mediated resistance to one ormore chemotherapeutic drugs; (ii) methods of treatment which cause anincrease in time of survival in patients with cancer; wherein the cancereither overexpresses thioredoxin or glutaredoxin and/or exhibits orpossesses thioredoxin- or glutaredoxin-mediated resistance to one ormore chemotherapeutic drugs; (iii) kits for the administration of thesecompositions to treat patients with cancer; (iv) methods forquantitatively ascertaining the level of expression of thioredoxin orglutaredoxin in patients with cancer; (v) methods of using the level andpattern of expression of thioredoxin or glutaredoxin in the cancer inthe initial diagnosis, planning of subsequent treatment methodologies,and/or ascertaining the potential treatment responsiveness of thespecific cancer of the patients with cancer; (vi) kits forquantitatively ascertaining the level of expression of thioredoxin orglutaredoxin in the cancer of patients with cancer; (vii) methods oftreatment which cause an increase in time of survival in patients withcancer; wherein the cancer either overexpresses thioredoxin orglutaredoxin and/or exhibits or possesses thioredoxin- orglutaredoxin-mediated resistance to one or more chemotherapeutic drugsand the treatment comprises the administration of the chemotherapeuticagents that are sensitive to thioredoxin and/or glutaredoxinoverexpression, either of which result in tumor mediated drug resistanceand enhanced angiogenesis; and (viii) methods for optimizing theschedule, dose, and combination of chemotherapy regimens in patients byascertaining, in-advance and throughout the treatment course, thethioredoxin levels, glutaredoxin levels and metabolic state in a samplefrom the patient with cancer.

In one embodiment of the present invention, a composition for increasingsurvival time in a patient with cancer is disclosed, wherein the cellscomprising the cancer which are isolated from the patient with cancereither: (i) overexpress thioredoxin or glutaredoxin and/or (ii) exhibitevidence of thioredoxin-mediated or glutaredoxin-mediated resistance tothe chemotherapeutic agent or agents used to treat said patient withcancer; is administered in a medically-sufficient dose to the patientwith cancer, either prior to, concomitantly with, or subsequent to theadministration of a chemotherapeutic agent or agents whose cytotoxic orcytostatic activity is adversely effected by either: (i) theoverexpression of thioredoxin or glutaredoxin and/or (ii) thethioredoxin-mediated or glutaredoxin-mediated treatment resistance.

It should be noted that the exhibition of thioredoxin-mediated orglutaredoxin-mediated treatment resistance is defined as “evidence of”due to the fact that it is neither expected, nor possible to prove with100% certainty that the cancer cells exhibit thioredoxin-mediated orglutaredoxin-mediated treatment resistance, prior to the treatment ofthe patient. By way of non-limiting example, the current use of, e.g.,florescence in situ hybridization (FISH) or immunohistochemistry (IHC)to guide treatment decisions for HER2/neu-based therapy are predicatedupon the probability of the overexpression/increased concentrations ofHER2/neu being correlated with the probability of a therapeuticresponse. Such expectation of a therapeutic response is not 100%certain, and is related to many factors, not the least of which is thediagnostic accuracy of the test utilized which, in turn, is also limitedby the sampling of the tumor and various other factors (e.g., laboratorymethodology/technique, reagent quality, and the like).

HER2/neu (also known as ErbB-2) is a protein which is associated with ahigher level of “aggressiveness” in breast cancers. HER2/neu is a memberof the ErbB protein family, more commonly known as the epidermal growthfactor receptor family (EGFR). It is a cell membrane surface-boundreceptor tyrosine kinase and is normally involved in the signaltransduction pathways leading to cell growth and differentiation. TheHER2 gene is a proto-oncogene located at the long arm of humanchromosome 17(17q11.2-q12). See, e.g., Olayioye, M. A., et al., Updateon HER-2 as a target for cancer therapy: intracellular signalingpathways of ErbB2/HER-2 and family members. Breast Cancer Res. 3:385-389(2001). HER2/neu plays an important role in the pathogenesis of breastcancer and serves as a target of treatment. Approximately 15-20 percentof breast cancers have an amplification of the HER2/neu gene oroverexpression of its protein product. Overexpression of HER2/neu inbreast cancer is associated with increased disease recurrence and worseprognosis. Overexpression of HER2/neu has also been shown to occur inother cancer, e.g., ovarian and stomach cancers. Clinically, HER2/neu isimportant as the target of the monoclonal antibody trastuzumab(Herceptin). Because of its prognostic role as well as its ability topredict response to trastuzumab, breast tumors are routinely checked foroverexpression of HER2/neu. Trastuzumab is only effective in breastcancer where the HER2/neu receptor is overexpressed. One of themechanisms of how traztuzumab works after it binds to HER2 is byincreasing p27, a protein that halts cell proliferation. See, e.g., Le,X. F., et al., HER2-targeting antibodies modulate the cyclin-dependentkinase inhibitor p27Kip1 via multiple signaling pathways. Cell Cycle 4:87-95 (2005). HER2 gene overexpression can be suppressed by theamplification of other genes and the use of the drug Herceptin.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of any cancerwhich either: (i) overexpresses thioredoxin or glutaredoxin and/or (ii)exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents being used to treatsaid patient with cancer.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of: lung cancer,colorectal cancer, gastric cancer, esophageal cancer, ovarian cancer,cancer of the biliary tract, gallbladder cancer, cervical cancer, breastcancer, endometrial cancer, vaginal cancer, prostate cancer, uterinecancer, hepatic cancer, pancreatic cancer, and adenocarcinoma.

In one embodiment of the present invention, a composition for increasingsurvival time in a patient with non-small cell lung carcinoma isdisclosed, wherein the non-small cell lung carcinoma, either: (i)overexpresses thioredoxin or glutaredoxin and/or (ii) exhibits evidenceof thioredoxin-mediated or glutaredoxin-mediated resistance to thechemotherapeutic agent or agents used to treat said patient withnon-small cell lung carcinoma; is administered in a medically-sufficientdose to the patient with non-small cell lung carcinoma, either prior to,concomitantly with, or subsequent to the administration of achemotherapeutic agent or agents whose cytotoxic or cytostatic activityis adversely affected by either: (i) the overexpression of thioredoxinor glutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance.

In another embodiment of the present invention, a composition forincreasing survival time in a patient with adenocarcinoma is disclosed,wherein the adenocarcinoma, either: (i) overexpresses thioredoxin orglutaredoxin and/or (ii) exhibits evidence of thioredoxin-mediated orglutaredoxin-mediated resistance to the chemotherapeutic agent or agentsused to treat said patient with adenocarcinoma; is administered in amedically-sufficient dose to the patient with adenocarcinoma, eitherprior to, concomitantly with, or subsequent to the administration of achemotherapeutic agent or agents whose cytotoxic or cytostatic activityis adversely affected by either: (i) the overexpression of thioredoxinor glutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance.

In another embodiment, the composition is a Formula (I) compound havingthe structural formula:

X—S—S—R₁-R₂:

wherein;

-   -   R₁ is a lower alkylene, wherein R₁ is optionally substituted by        a member of the group consisting of: lower alkyl, aryl, hydroxy,        alkoxy, aryloxy, mercapto,    -   alkylthio or arylthio, for a corresponding hydrogen atom, or

-   -   R₂ and R₄ is sulfonate or phosphonate;    -   R₅ is hydrogen, hydroxy, or sulfhydryl;    -   m is 0, 1, 2, 3, 4, 5, or 6; and    -   X is a sulfur-containing amino acid or a peptide consisting of        from 2-10 amino acids;    -   or wherein X is a member of the group consisting of: lower        thioalkyl (lower mercapto alkyl), lower alkylsulfonate, lower        alkylphosphonate, lower alkenylsulfonate, lower alkyl, lower        alkenyl, lower alkynyl, aryl, alkoxy, aryloxy, mercapto,        alkylthio or hydroxy for a corresponding hydrogen atom; and        pharmaceutically-acceptable salts, prodrugs, analogs,        conjugates, hydrates, solvates, polymorphs, stereoisomers        (including diastereoisomers and enantiomers) and tautomers        thereof.

In one embodiment of the present invention, the composition is apharmaceutically-acceptable disodium salt of a Formula (I) compound. Invarious other embodiments, the composition of the present inventionis/are a pharmaceutically-acceptable salt(s) of a Formula (I) compoundwhich include, for example: (i) a monosodium salt; (ii) a sodiumpotassium salt; (iii) a dipotassium salt; (iv) a calcium salt; (v) amagnesium salt; (vi) a manganese salt; (vii) a monopotassium salt; and(viii) an ammonium salt. It should be noted that mono- and di-potassiumsalts of 2,2′-dithio-bis-ethane sulfonate and/or an analog thereof areadministered to a subject if the total dose of potassium administered atany given point in time is not greater than 100 Meq. and the subject isnot hyperkalemic and does not have a condition that would predispose thesubject to hyperkalemia (e.g., renal failure).

In another embodiment of the present invention, the composition isdisodium 2,2′-dithio-bis-ethane sulfonate (also known in the literatureas Tavocept™, BNP7787, and dimesna).

In yet another embodiment, the composition is 2-mercapto-ethanesulfonate or 2-mercapto-ethane sulfonate conjugated as a disulfide witha substituent group selected from the group consisting of:

-Cys, -Homocysteine, -Cys-Gly, -Cys-Glu, -Homocysteine,-Homocysteine-Gly, -Homocysteine-Glu, -Cys-Glu,

wherein R₁ and R₂ are any L- or D-amino acids.

In another embodiment, the chemotherapy agent or agents administered areselected from the group consisting of fluoropyrimidines; pyrimidinenucleosides; purine nucleosides; anti-folates, platinum agents;anthracyclines/anthracenediones; epipodophyllotoxins; camptothecins;hormones; hormonal complexes; antihormonals; enzymes, proteins, peptidesand polyclonal and/or monoclonal antibodies; vinca alkaloids; taxanes;epothilones; antimicrotubule agents; alkylating agents; antimetabolites;topoisomerase inhibitors; aziridine-containing compounds; antivirals;and various other cytotoxic and cytostatic agents.

In one embodiment of the present invention, the chemotherapy agent oragents are selected from the group consisting of: cisplatin,carboplatin, oxaliplatin, satraplatin, picoplatin, tetraplatin,platinum-DACH, and analogs and derivatives thereof.

In another embodiment, the chemotherapy agent or agents are selectedfrom the group consisting of: docetaxel, paclitaxel, polyglutamylatedforms of paclitaxel, liposomal paclitaxel, and analogs and derivativesthereof.

In yet another embodiment of the present invention, the chemotherapyagents are docetaxel and cisplatin.

The present invention additionally involves the use of the methods andthe administration of the compositions described herein to a subject,optionally with or within a device, wherein the administration takesplace as medically indicated in the subject prior to, concurrently orsimultaneously, or following the administration of any chemotherapeuticagent or pharmaceutically active compound(s) by any route, dose,concentration, osmolarity, duration or schedule. Some of such routes,doses, concentrations, osmolarities, durations or schedules have beendisclosed in U.S. patent application Ser. No. 11/638,193, entitled“CHEMOPROTECTIVE METHODS AND COMPOSITIONS”, filed Dec. 13, 2006, thedisclosure of which is hereby incorporated by reference in its entirety.Embodiments of the present invention also include controlled or otherdoses, dosage forms, formulations, compositions and/or devicescontaining one or more chemotherapeutic agents and a Formula (I)compound of the present invention, which include 2,2′-dithio-bis-ethanesulfonate, a pharmaceutically-acceptable salt, an analog thereof; mesna,a mesna heteroconjugate; and the various other Formula (I) compounds,including doses and dosage forms for: (i) oral (e.g., tablet,suspension, solution, gelatin capsule (hard or soft), sublingual,dissolvable tablet, troche, and the like); (ii) injection (e.g.,subcutaneous administration, intradermal administration, subdermaladministration, intramuscular administration, depot administration,intravenous administration, intra-arterial administration, and thelike); (iii) intra-cavitary (e.g., into the intrapleural,intraperitoneal, intravesicular, and/or intrathecal spaces); (iv) perrectum (e.g., suppository, retention enema); and (v) topicaladministration routes.

Various chemotherapeutic agents may be used in conjunction with, or as apart of, the compositions, methods, and kits described and claimedherein. Chemotherapeutic agents may include, for example, afluoropyrimidine; a pyrimidine nucleoside; a purine nucleoside; anantifolate, a platinum analog; an anthracycline/anthracenedione; anepipodophyllotoxin; a camptothecin; a hormone; a hormonal analog; anantihormonal; an enzyme, protein, peptide, or polyclonal or monoclonalantibody; a vinca alkaloid; a taxane; an epothilone; an antimicrotubuleagent; an alkylating agent; an antimetabolite; a topoisomeraseinhibitor; an aziridine-containing compound; an antiviral; or anothercytotoxic and/or cytostatic agent.

More specifically, fluoropyrimidines include, for example,5-fluorouracil (5-FU), S-1, capecitabine, ftorafur, 5′deoxyfluorouridine, UFT, eniluracil, and the like. Pyrimidinenucleosides include, for example, cytarabine, deoxycytidine,5-azacytosine, gemcitabine, 5-azadeoxycytidine, and the like. Purinenucleosides include, for example, fludarabine, 6-mercaptopurine,thioguanine, allopurinol, cladribine, and 2-chloro adenosine.Antifolates include, for example, methotrexate (MTX), pemetrexed(Alimta®), trimetrexate, aminopterin, methylene-10-deazaminopterin(MDAM), and the like. Platinum analogs include those in which theplatinum moiety can have a valence of II or IV and specifically include,for example, cisplatin, carboplatin, oxaliplatin, satraplatin,picoplatin, tetraplatin, platinum-DACH, and analogs thereof. Taxanemedicaments include, for example, docetaxel or paclitaxel (including thecommercially-available paclitaxel derivatives Taxol® and Abraxane®),polyglutamylated forms of paclitaxel (e.g., Xyotax®), liposomalpaclitaxel (e.g., Tocosol®), and analogs and derivatives thereof.Anthracyclines/anthracenediones include, for example, doxorubicin,daunorubicin, epirubicin, and idarubicin. Epipodophyllotoxin derivativesinclude, for example, etoposide, etoposide phosphate and teniposide.Camptothecins include, for example, irinotecan, topotecan,9-aminocamptothecin, 10,11-methylenedioxycamptothecin, karenitecin,9-nitrocamptothecin, and TAS 103. Hormones and hormonal analogs mayinclude, for example, (i) estrogens and estrogen analogs, includinganastrazole, diethylstilbesterol, estradiol, premarin, raloxifene;progesterone, progesterone analogs and progestins, includingprogesterone, norethynodrel, esthisterone, dimesthisterone, megestrolacetate, medroxyprogesterone acetate, hydroxyprogesterone caproate, andnorethisterone; (ii) androgens, including fluoxymesterone,methyltestosterone and testosterone; and (iii) adrenocorticosteroids,including dexamthasone, prednisone, cortisol, solumedrol, and the like.Antihormones include, for example, (i) antiestrogens, including:tamoxifen, fulvestrant, toremifene; aminoglutethimide, testolactone,droloxifene, and anastrozole; (ii) antiandrogens, including:bicalutamide, flutamide, nilutamide, and goserelin; (iii)antitestosterones, including: flutamide, leuprolide, and triptorelin;and (iv) adrenal steroid inhibitors including: aminoglutethimide andmitotane; and anti-leuteinizing hormones, including goserelin. Enzymes,proteins, peptides, polyclonal and/or monoclonal antibodies, mayinclude, for example, asparaginase, cetuximab, erlotinib, bevacizumab,rituximab, gefitinib, trastuzumab, interleukins, interferons,leuprolide, pegasparaginase, and the like. Vinca Alkaloids include, forexample, vincristine, vinblastine, vinorelbine, vindesine, and the like.Alkylating agents may include, for example, dacarbazine; procarbazine;temozolamide; thiotepa, nitrogen mustards (e.g., mechlorethamine,chlorambucil, L-phenylalanine mustard, melphalan, and the like);oxazaphosphorines (e.g., ifosphamide, cyclophosphamide, mefosphamide,perfosfamide, trophosphamide and the like); alkyl sulfonates (e.g.,busulfan); and nitrosoureas (e.g., carmustine, lomustine, semustine, andthe like). Epothilones include, for example, epothilones A-E.Antimetabolites include, for example, tomudex and methotrexate,trimetrexate, aminopterin, pemetrexid, MDAM, 6-mercaptopurine, and6-thioguanine. Topoisomerase inhibitors include, for example,irinotecan, topotecan, karenitecin, amsacrine, etoposide, etoposidephosphate, teniposide, and doxorubicin, daunorubicin, and other analogs.Antiviral agents include, for example, acyclovir, valacyclovir,ganciclovir, amantadine, rimantadine, lamivudine, and zidovudine.Monoclonal antibody agents include, for example, bevacizumab,trastuzumab, rituximab, and the like, as well as growth inhibitors suchas erlotinib, and the like. In general, cytostatic agents aremechanism-based agents that slow the progression of neoplastic disease.

Chemotherapeutic agents may be prepared and administered to subjectsusing methods known within the art. For example, paclitaxel may beprepared using methods described in U.S. Pat. Nos. 5,641,803, 6,506,405,and 6,753,006 and is administered as known in the art (see, e.g., U.S.Pat. Nos. 5,641,803, 6,506,405, and 6,753,006). Paclitaxel may beprepared for administration in a dose in the range of approximately 50mg/m² and approximately 275 mg/m². Preferred doses include approximately80 mg/m², approximately 135 mg/m² and approximately 175 mg/m².

Docetaxel may be prepared using methods described in U.S. Pat. No.4,814,470 and is administered as known in the art (see, e.g., U.S. Pat.Nos., 4,814,470, 5,438,072, 5,698,582, and 5,714,512). Docetaxel may beprepared for administration in a dose in the range of approximately 30mg/m² to approximately 100 mg/m². Preferred doses include approximately55 mg/m², approximately 60 mg/m², approximately 75 mg/m², andapproximately 100 mg/m².

Cisplatin may be prepared using methods described in U.S. Pat. Nos.4,302,446, 4,322,391, 4,310,515, and 4,915,956 and is administered asknown in the art (see, e.g., U.S. Pat. Nos. 4,177,263, 4,310,515,4,451,447). Cisplatin may be prepared for administration in a dose inthe range of approximately 30 mg/m² to approximately 120 mg/m² in asingle dose or 15 mg/m² to approximately 20 mg/m² daily for five days.Preferred doses include approximately 50 mg/m², approximately 75 mg/m²and approximately 100 mg/m².

Carboplatin may be prepared using methods described in U.S. Pat. No.4,657,927 and is administered as known in the art (see, e.g., U.S. Pat.No. 4,657,927). Carboplatin may be prepared for administration in a dosein the range of approximately 20 mg/kg to approximately 200 mg/kg.Preferred doses include approximately 300 mg/m² and approximately 360mg/m². Other dosing may be calculated using a formula according to themanufacturer's instructions.

Oxaliplatin may be prepared using methods described in U.S. Pat. Nos.5,290,961, 5,420,319, 5,338,874 and is administered as known in the art(see, e.g., U.S. Pat. No. 5,716,988). Oxaliplatin may be prepared foradministration in a dose in the range of approximately 50 mg/m² toapproximately 200 mg/m². Preferred doses include approximately 85 mg/m²and approximately 130 mg/m².

In one embodiment of the present invention, a method of increasingsurvival time in a patient with cancer is disclosed, wherein the cancer,either: (i) overexpresses thioredoxin or glutaredoxin and/or (ii)exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents used to treat saidpatient with non-small cell lung carcinoma; wherein said methodcomprises the administration of a medically-sufficient dose of a Formula(I) compound to said patient with cancer either prior to, concomitantlywith, or subsequent to the administration of a chemotherapeutic agent oragents whose cytotoxic or cytostatic activity is adversely affected byeither: (i) the overexpression of thioredoxin or glutaredoxin and/or(ii) the thioredoxin-mediated or glutaredoxin-mediated treatmentresistance.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of any cancerwhich either: (i) overexpresses thioredoxin or glutaredoxin and/or (ii)exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents being used to treatsaid patient with cancer.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of: lung cancer,colorectal cancer, gastric cancer, esophageal cancer, ovarian cancer,cancer of the biliary tract, gallbladder cancer, cervical cancer, breastcancer, endometrial cancer, vaginal cancer, prostate cancer, uterinecancer, hepatic cancer, pancreatic cancer, and adenocarcinoma.

In another embodiment of the present invention, a method of increasingsurvival time in a patient with non-small cell lung carcinoma isdisclosed, wherein the non-small lung carcinoma, either: (i)overexpresses thioredoxin or glutaredoxin and/or (ii) exhibits evidenceof thioredoxin-mediated or glutaredoxin-mediated resistance to thechemotherapeutic agent or agents used to treat said patient withnon-small cell lung carcinoma; wherein said method comprises theadministration of a medically-sufficient dose of a Formula (I) compoundto said patient with non-small cell lung carcinoma either prior to,concomitantly with, or subsequent to the administration of achemotherapeutic agent or agents whose cytotoxic or cytostatic activityis adversely affected by either: (i) the overexpression of thioredoxinor glutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance.

In yet another embodiment of the present invention, a method ofincreasing survival time in a patient with adenocarcinoma is disclosed,wherein the adenocarcinoma, either: (i) overexpresses thioredoxin orglutaredoxin and/or (ii) exhibits evidence of thioredoxin-mediated orglutaredoxin-mediated resistance to the chemotherapeutic agent or agentsused to treat said patient with adenocarcinoma; wherein said methodcomprises the administration of a medically-sufficient dose of a Formula(I) compound to said patient with adenocarcinoma either prior to,concomitantly with, or subsequent to the administration of achemotherapeutic agent or agents whose cytotoxic or cytostatic activityis adversely affected by either: (i) the overexpression of thioredoxinor glutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance.

In one embodiment, the Formula (I) compound has the structural formula:

X—S—S—R₁-R₂:

wherein;

-   -   R₁ is a lower alkylene, wherein R₁ is optionally substituted by        a member of the group consisting of: lower alkyl, aryl, hydroxy,        alkoxy, aryloxy, mercapto,    -   alkylthio or arylthio, for a corresponding hydrogen atom, or

-   -   R₂ and R₄ is sulfonate or phosphonate;    -   R₅ is hydrogen, hydroxy, or sulfhydryl;    -   m is 0, 1, 2, 3, 4, 5, or 6; and    -   X is a sulfur-containing amino acid or a peptide consisting of        from 2-10 amino acids;    -   or wherein X is a member of the group consisting of: lower        thioalkyl (lower mercapto alkyl), lower alkylsulfonate, lower        alkylphosphonate, lower alkenylsulfonate, lower alkyl, lower        alkenyl, lower alkynyl, aryl, alkoxy, aryloxy, mercapto,        alkylthio or hydroxy for a corresponding hydrogen atom; and        pharmaceutically-acceptable salts, prodrugs, analogs,        conjugates, hydrates, solvates, polymorphs, stereoisomers        (including diastereoisomers and enantiomers) and tautomers        thereof.

In one embodiment of the present invention, the composition is apharmaceutically-acceptable disodium salt of a Formula (I) compound. Invarious other embodiments, the composition of the present inventionis/are a pharmaceutically-acceptable salt(s) of a Formula (I) compoundwhich include, for example: (i) a monosodium salt; (ii) a sodiumpotassium salt; (iii) a dipotassium salt; (iv) a calcium salt; (v) amagnesium salt; (vi) a manganese salt; (vii) a monopotassium salt; and(viii) an ammonium salt. It should be noted that mono- and di-potassiumsalts of 2,2′-dithio-bis-ethane sulfonate and/or an analog thereof areadministered to a subject if the total dose of potassium administered atany given point in time is not greater than 100 Meq. and the subject isnot hyperkalemic and does not have a condition that would predispose thesubject to hyperkalemia (e.g., renal failure).

In another embodiment of the present invention, the composition isdisodium 2,2′-dithio-bis-ethane sulfonate (also known in the literatureas Tavocept™, BNP7787, and dimesna).

In yet another embodiment, the composition is 2-mercapto-ethanesulfonate or 2-mercapto-ethane sulfonate conjugated as a disulfide witha substituent group selected from the group consisting of:

-Cys, -Homocysteine, -Cys-Gly, -Cys-Glu, -Homocysteine,-Homocysteine-Gly, -Homocysteine-Glu, -Cys-Glu,

wherein R₁ and R₂ are any L- or D-amino acids.

In another embodiment, the chemotherapy agent or agents administered areselected from the group consisting of fluoropyrimidines; pyrimidinenucleosides; purine nucleosides;

anti-folates, platinum agents; anthracyclines/anthracenediones;epipodophyllotoxins; camptothecins; hormones; hormonal complexes;antihormonals; enzymes, proteins, peptides and polyclonal and/ormonoclonal antibodies; vinca alkaloids; taxanes; epothilones;antimicrotubule agents; alkylating agents; antimetabolites;topoisomerase inhibitors; aziridine-containing compounds; antivirals;and various other cytotoxic and cytostatic agents.

In one embodiment of the present invention, the chemotherapy agent oragents are selected from the group consisting of: cisplatin,carboplatin, oxaliplatin, satraplatin, picoplatin, tetraplatin,platinum-DACH, and analogs and derivatives thereof.

In another embodiment, the chemotherapy agent or agents are selectedfrom the group consisting of: docetaxel, paclitaxel, polyglutamylatedforms of paclitaxel, liposomal paclitaxel, and analogs and derivativesthereof.

In yet another embodiment of the present invention, the chemotherapyagents are docetaxel and cisplatin.

In one embodiment of the present invention, a kit comprising a Formula(I) compound for administration, and instructions for administering saidFormula (I) compound to a patient with cancer in an amount sufficient tocause an increase in the survival time of said patient with cancer whois receiving a chemotherapeutic agent or agents whose cytotoxic orcytostatic activity is adversely affected by either: (i) theoverexpression of thioredoxin or glutaredoxin and/or (ii) thethioredoxin-mediated or glutaredoxin-mediated treatment resistance, isdisclosed.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of any cancerwhich either: (i) overexpresses thioredoxin or glutaredoxin and/or (ii)exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents being used to treatsaid patient with cancer.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of lung cancer,colorectal cancer, gastric cancer, esophageal cancer, ovarian cancer,cancer of the biliary tract, gallbladder cancer, cervical cancer, breastcancer, endometrial cancer, vaginal cancer, prostate cancer, uterinecancer, hepatic cancer, pancreatic cancer, and adenocarcinoma.

In still another embodiment, the Formula (I) compound has the structuralformula:

X—S—S—R₁-R₂:

wherein;

-   -   R₁ is a lower alkylene, wherein R₁ is optionally substituted by        a member of the group consisting of: lower alkyl, aryl, hydroxy,        alkoxy, aryloxy, mercapto, alkylthio or arylthio, for a        corresponding hydrogen atom, or

-   -   R₂ and R₄ is sulfonate or phosphonate;    -   R₅ is hydrogen, hydroxy, or sulfhydryl;    -   m is 0, 1, 2, 3, 4, 5, or 6; and    -   X is a sulfur-containing amino acid or a peptide consisting of        from 2-10 amino acids;    -   or wherein X is a member of the group consisting of: lower        thioalkyl (lower mercapto alkyl), lower alkylsulfonate, lower        alkylphosphonate, lower alkenylsulfonate, lower alkyl, lower        alkenyl, lower alkynyl, aryl, alkoxy, aryloxy, mercapto,        alkylthio or hydroxy for a corresponding hydrogen atom; and        pharmaceutically-acceptable salts, prodrugs, analogs,        conjugates, hydrates, solvates, polymorphs, stereoisomers        (including diastereoisomers and enantiomers) and tautomers        thereof.

In one embodiment of the present invention, the Formula (I) compound isselected from the group consisting of: a disodium salt, a monosodiumsalt, a sodium potassium salt, a dipotassium salt, a monopotassium salt,a calcium salt, a magnesium salt, an ammonium salt, or a manganese salt.

In another embodiment, the Formula (I) compound is a disodium salt.

In yet another embodiment, the Formula (I) compound is disodium2,2′-dithio-bis-ethane sulfonate.

In yet another embodiment, the composition is 2-mercapto-ethanesulfonate or 2-mercapto-ethane sulfonate conjugated as a disulfide witha substituent group selected from the group consisting of:

-Cys, -Homocysteine, -Cys-Gly, -Cys-Glu, -Homocysteine,-Homocysteine-Gly, -Homocysteine-Glu, -Cys-Glu,

wherein R₁ and R₂ are any L- or D-amino acids.

In one embodiment, the chemotherapy agent or agents are selected fromthe group consisting of: fluoropyrimidines; pyrimidine nucleosides;purine nucleosides; anti-folates, platinum agents;anthracyclines/anthracenediones; epipodophyllotoxins; camptothecins;hormones; hormonal complexes; antihormonals; enzymes, proteins, peptidesand polyclonal and/or monoclonal antibodies; vinca alkaloids; taxanes;epothilones; antimicrotubule agents; alkylating agents; antimetabolites;topoisomerase inhibitors; aziridine-containing compounds; antivirals;and various other cytotoxic and cytostatic agents.

In another embodiment, the chemotherapy agent or agents are selectedfrom the group consisting of: cisplatin, carboplatin, oxaliplatin,satraplatin, picoplatin, tetraplatin, platinum-DACH, and analogs andderivatives thereof.

In still another embodiment of the present invention, the chemotherapyagent or agents are selected from the group consisting of docetaxel,paclitaxel, polyglutamylated forms of paclitaxel, liposomal paclitaxel,and analogs and derivatives thereof.

In one embodiment, the chemotherapy agents are docetaxel and cisplatin.

In another embodiment of the present invention, a kit comprising aFormula (I) compound for administration, and instructions foradministering said Formula (I) compound to a patient with non-small celllung carcinoma in an amount sufficient to cause an increase in thesurvival time of said patient who is receiving a chemotherapeutic agentor agents whose cytotoxic or cytostatic activity is adversely affectedby either: (i) the overexpression of thioredoxin or glutaredoxin and/or(ii) the thioredoxin-mediated or glutaredoxin-mediated treatmentresistance, is disclosed.

In yet another embodiment, a kit comprising a Formula (I) compound foradministration, and instructions for administering said Formula (I)compound to a patient with adenocarcinoma in an amount sufficient tocause an increase in the survival time of said patient who is receivinga chemotherapeutic agent or agents whose cytotoxic or cytostaticactivity is adversely affected by either: (i) the overexpression ofthioredoxin or glutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance, is disclosed.

In one embodiment, the Formula (I) compound has the structural formula:

—S—S—R₁-R₂:

wherein;

-   -   R₁ is a lower alkylene, wherein R₁ is optionally substituted by        a member of the group consisting of lower alkyl, aryl, hydroxy,        alkoxy, aryloxy, mercapto, alkylthio or arylthio, for a        corresponding hydrogen atom, or

-   -   R₂ and R₄ is sulfonate or phosphonate;    -   R₅ is hydrogen, hydroxy, or sulfhydryl;    -   m is 0, 1, 2, 3, 4, 5, or 6; and    -   X is a sulfur-containing amino acid or a peptide consisting of        from 2-10 amino acids;    -   or wherein X is a member of the group consisting of: lower        thioalkyl (lower mercapto alkyl), lower alkylsulfonate, lower        alkylphosphonate, lower alkenylsulfonate, lower alkyl, lower        alkenyl, lower alkynyl, aryl, alkoxy, aryloxy, mercapto,        alkylthio or hydroxy for a corresponding hydrogen atom; and        pharmaceutically-acceptable salts, prodrugs, analogs,        conjugates, hydrates, solvates, polymorphs, stereoisomers        (including diastereoisomers and enantiomers) and tautomers        thereof.

In one embodiment of the present invention, the composition is apharmaceutically-acceptable disodium salt of a Formula (I) compound. Invarious other embodiments, the composition of the present inventionis/are a pharmaceutically-acceptable salt(s) of a Formula (I) compoundwhich include, for example: (i) a monosodium salt; (ii) a sodiumpotassium salt; (iii) a dipotassium salt; (iv) a calcium salt; (v) amagnesium salt; (vi) a manganese salt; (vii) a monopotassium salt; and(viii) an ammonium salt. It should be noted that mono- and di-potassiumsalts of 2,2′-dithio-bis-ethane sulfonate and/or an analog thereof areadministered to a subject if the total dose of potassium administered atany given point in time is not greater than 100 Meq. and the subject isnot hyperkalemic and does not have a condition that would predispose thesubject to hyperkalemia (e.g., renal failure).

In another embodiment of the present invention, the composition isdisodium 2,2′-dithio-bis-ethane sulfonate (also known in the literatureas Tavocept™, BNP7787, and dimesna).

In yet another embodiment, the composition is 2-mercapto-ethanesulfonate or 2-mercapto-ethane sulfonate conjugated as a disulfide witha substituent group selected from the group consisting of:

-Cys, -Homocysteine, -Cys-Gly, -Cys-Glu, -Homocysteine,-Homocysteine-Gly, -Homocysteine-Glu, -Cys-Glu,

wherein R₁ and R₂ are any L- or D-amino acids.

In another embodiment, the chemotherapy agent or agents administered areselected from the group consisting of fluoropyrimidines; pyrimidinenucleosides; purine nucleosides; anti-folates, platinum agents;anthracyclines/anthracenediones; epipodophyllotoxins; camptothecins;hormones; hormonal complexes; antihormonals; enzymes, proteins, peptidesand polyclonal and/or monoclonal antibodies; vinca alkaloids; taxanes;epothilones; antimicrotubule agents; alkylating agents; antimetabolites;topoisomerase inhibitors; aziridine-containing compounds; antivirals;and various other cytotoxic and cytostatic agents.

In one embodiment of the present invention, the chemotherapy agent oragents are selected from the group consisting of cisplatin, carboplatin,oxaliplatin, satraplatin, picoplatin, tetraplatin, platinum-DACH, andanalogs and derivatives thereof.

In another embodiment, the chemotherapy agent or agents are selectedfrom the group consisting of: docetaxel, paclitaxel, polyglutamylatedforms of paclitaxel, liposomal paclitaxel, and analogs and derivativesthereof.

In yet another embodiment of the present invention, the chemotherapyagents are docetaxel and cisplatin.

In one embodiment of the present invention, a method for quantitativelyascertaining the level of thioredoxin or glutaredoxin DNA, mRNA, orprotein in cells which have been isolated from a patient who issuspected of having cancer or has already been diagnosed with cancer isdisclosed; wherein the method used to identify levels of thioredoxin orglutaredoxin is selected from the group consisting of: fluorescence insitu hybridization (FISH), nucleic acid microarray analysis,immunohistochemistry (IHC), and radioimmunoassay (RIA).

In another embodiment, the method is used in the initial diagnosis, theplanning of subsequent treatment methodologies, and/or determining thepotential aggressiveness of cancer growth in a patient suffering from atype of cancer in which the cells comprising the cancer either: (i)overexpress thioredoxin or glutaredoxin and/or (ii) exhibit evidence ofthioredoxin-mediated or glutaredoxin-mediated treatment resistance tothe chemotherapeutic agents or agents already being administered to thepatient with cancer.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of any cancerwhich either: (i) overexpresses thioredoxin or glutaredoxin and/or (ii)exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents being used to treatsaid patient with cancer.

In still another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of lung cancer,colorectal cancer, gastric cancer, esophageal cancer, ovarian cancer,cancer of the biliary tract, gallbladder cancer, cervical cancer, breastcancer, endometrial cancer, vaginal cancer, prostate cancer, uterinecancer, hepatic cancer, pancreatic cancer, and adenocarcinoma.

In another embodiment of the present invention, a method for increasingsurvival time in a patient with cancer is disclosed, wherein saidcancer, either: (i) overexpresses thioredoxin or glutaredoxin and/or(ii) exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents used to treat saidpatient with cancer; wherein said method comprises the administration ofa medically-sufficient dose of a Formula (I) compound to said patientwith cancer either prior to, concomitantly with, or subsequent to theadministration of the chemotherapeutic agents cisplatin and docetaxel;wherein the cytotoxic or cytostatic activity of the chemotherapeuticagents is adversely affected by either: (i) the overexpression ofthioredoxin or glutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of any cancerwhich either: (i) overexpresses thioredoxin or glutaredoxin and/or (ii)exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents being used to treatsaid patient with cancer.

In another embodiment, the cancer of origin for treatment with thepresent invention is selected from the group consisting of lung cancer,colorectal cancer, gastric cancer, esophageal cancer, ovarian cancer,cancer of the biliary tract, gallbladder cancer, cervical cancer, breastcancer, endometrial cancer, vaginal cancer, prostate cancer, uterinecancer, hepatic cancer, pancreatic cancer, and adenocarcinoma.

In one embodiment of the present invention, a method for increasingsurvival time in a cancer patient with non-small cell lung carcinoma isdisclosed, wherein the non-small cell lung carcinoma, either: (i)overexpresses thioredoxin or glutaredoxin and/or (ii) exhibits evidenceof thioredoxin-mediated or glutaredoxin-mediated resistance to thechemotherapeutic agent or agents used to treat said patient withnon-small cell lung carcinoma; wherein said method comprises theadministration of a medically-sufficient dose of a Formula (I) compoundto said patient with non-small cell lung carcinoma either prior to,concomitantly with, or subsequent to the administration of thechemotherapeutic agents cisplatin and docetaxel; wherein the cytotoxicor cytostatic activity of said chemotherapeutic agents is adverselyaffected by either: (i) the overexpression of thioredoxin orglutaredoxin and/or (ii) the thioredoxin-mediated orglutaredoxin-mediated treatment resistance.

In another embodiment, a method for increasing survival time in a cancerpatient with adenocarcinoma is disclosed, wherein the adenocarcinoma,either: (i) overexpresses thioredoxin or glutaredoxin and/or (ii)exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents used to treat saidpatient with adenocarcinoma; wherein said method comprises theadministration of a medically-sufficient dose of a Formula (I) compoundto said patient with adenocarcinoma either prior to, concomitantly with,or subsequent to the administration of the chemotherapeutic agentscisplatin and docetaxel; wherein the cytotoxic or cytostatic activity ofsaid chemotherapeutic agents is adversely affected by either: (i) theoverexpression of thioredoxin or glutaredoxin and/or (ii) thethioredoxin-mediated or glutaredoxin-mediated treatment resistance.

In yet another embodiment, the method is comprised of: (i) theadministration of docetaxel at a dose of 75 mg/m² which is givenintravenously over a period of approximately 1 hour; (ii) theadministration of docetaxel in step (i) is immediately followed by theadministration of disodium 2,2′-dithio-bis-ethane sulfonate (Tavocept™)at a dose of approximately 40 grams which is given intravenously over aperiod of approximately 30 minutes; and (iii) the administration ofdisodium 2,2′-dithio-bis-ethane sulfonate (Tavocept™) in step (ii) isimmediately followed by the administration of cisplatin at a dose of 75mg/m² which is given intravenously over a period of approximately 1 hourwith concomitant sufficient intravenous hydration; wherein steps(i)-(iii) constitute a single chemotherapy cycle which can be repeatedevery two weeks, for up to a total of six cycles.

In another embodiment, a kit comprising a Formula (I) compound foradministration, and instructions for administering said Formula (I)compound to a patient with any medical condition or disease whereinthere is overexpression of thioredoxin or glutaredoxin is disclosed,wherein said kit comprises the administration of a medically-sufficientdose of a Formula (I) compound overexpression, and wherein theoverexpression of thioredoxin or glutaredoxin causes deleteriousphysiological effects in said patient.

Furthermore, in brief, the present invention discloses and claims: (i)compositions, methods, and kits which lead to an increase in patientsurvival time in cancer patients receiving chemotherapy; (ii)compositions and methods which cause cytotoxic or apoptotic potentiationof the anti-cancer activity of chemotherapeutic agents; (iii)compositions and methods for maintaining or stimulating hematologicalfunction in patients in need thereof, including those patients sufferingfrom cancer; (iv) compositions and methods for maintaining orstimulating erythropoietin function or synthesis in patients in needthereof, including those patients suffering from cancer; (v)compositions and methods for mitigating or preventing anemia in patientsin need thereof, including those patients suffering from cancer; (vi)compositions and methods for maintaining or stimulating pluripotent,multipotent, and unipotent normal stem cell function or synthesis inpatients in need thereof, including those patients suffering fromcancer; (vii) compositions and methods which promote the arrest orretardation of tumor progression in those cancer patients receivingchemotherapy; (viii) compositions and methods for increasing patientsurvival and/or delaying tumor progression while maintaining orimproving the quality of life in a cancer patient receivingchemotherapy; (ix) novel methods of the administration of taxane and/orplatinum medicaments and a Formula (I) compound of the present inventionto a cancer patient; and (x) kits to achieve one or more of theaforementioned physiological effects in a patient in need thereof,including those patients suffering from cancer.

In one embodiment, a patient suffering from lung cancer treated withtaxane and/or platinum medicaments is given a medically sufficientdosage of a Formula (I) compound so as to increase patient survival timein said patient suffering from lung cancer.

In another embodiment, the lung cancer is non-small cell lung carcinoma.

In another embodiment, the increase in patient survival time in saidpatient suffering from lung cancer and treated with a Formula (I)compound is expected to be at least 30 days longer than the expectedsurvival time if said patient was not treated with a Formula (I)compound.

In yet another embodiment, a patient suffering from lung cancer wastreated with paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks, wherein the dose of paclitaxel ranged fromapproximately 160 mg/m² to approximately 190 mg/m², the dose of aFormula (I) compound ranged from approximately 14 g/m² to approximately22 g/m², and the dose of cisplatin ranged from approximately 60 mg/m² toapproximately 100 mg/m², wherein said administration of paclitaxel, aFormula (I) compound, and cisplatin once every 2-4 weeks was repeated atleast once.

In still another embodiment, a patient suffering from lung cancer wastreated with paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks, wherein the dose of paclitaxel was approximately 175mg/m², the dose of a Formula (I) compound was approximately 18.4 g/m²,and the dose of cisplatin ranged from approximately 75 mg/m² toapproximately 85 mg/m², wherein said administration of paclitaxel, aFormula (I) compound, and cisplatin once every 3 weeks was repeated for6 cycles.

In another embodiment, the patients suffering from lung cancer were maleor female and smokers or non-smokers.

In one embodiment, a patient suffering from adenocarcinoma treated withtaxane and/or platinum medicaments is given a medically sufficientdosage of a Formula (I) compound so as to increase patient survival timein said patient suffering from adenocarcinoma.

In another embodiment, the increase in patient survival time in saidpatient suffering from adenocarcinoma and treated with a Formula (I)compound is expected to be at least 30 days longer than the expectedsurvival time if said patient was not treated with a Formula (I)compound.

In yet another embodiment, a patient suffering from adenocarcinoma istreated with paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks, wherein the dose of paclitaxel ranged fromapproximately 160 mg/m² to approximately 190 mg/m², the dose of aFormula (I) compound ranged from approximately 14 g/m² to approximately22 g/m², and the dose of cisplatin ranged from approximately 60 mg/m² toapproximately 100 mg/m², wherein said administration of paclitaxel, aFormula (I) compound, and cisplatin once every 2-4 weeks was repeated atleast once.

In still another embodiment, a patient suffering from adenocarcinoma istreated with paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks, wherein the dose of paclitaxel was approximately 175mg/m², the dose of a Formula (I) compound was approximately 18.4 g/m²,and the dose of cisplatin ranged from approximately 75 mg/m² toapproximately 85 mg/m², wherein said administration of paclitaxel, aFormula (I) compound, and cisplatin once every 3 weeks was repeated for6 cycles.

In another embodiment, the patients suffering from adenocarcinoma weremale or female and smokers or non-smokers.

In one embodiment, a patient suffering from lung cancer treated withtaxane and platinum medicaments is given a medically sufficient dosageof a Formula (I) compound so as to potentiate the chemotherapeuticeffect in said patient suffering from lung cancer.

In another embodiment, the lung cancer is non-small cell lung carcinoma.

In yet another embodiment, the chemotherapeutic effect is potentiated ina patient suffering from lung cancer treated with paclitaxel, a Formula(I) compound, and cisplatin once every 2-4 weeks, wherein the dose ofpaclitaxel ranged from approximately 160 mg/m² to approximately 190mg/m², the dose of a Formula (I) compound ranged from approximately 14g/m² to approximately 22 g/m², and the dose of cisplatin ranged fromapproximately 60 mg/m² to approximately 100 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks was repeated at least once.

In still another embodiment, the chemotherapeutic effect is potentiatedin a patient suffering from lung cancer treated with paclitaxel, aFormula (I) compound, and cisplatin once every 3 weeks, wherein the doseof paclitaxel was approximately 175 mg/m², the dose of a Formula (I)compound was approximately 18.4 g/m², and the dose of cisplatin rangedfrom approximately 75 mg/m² to approximately 85 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from lung cancer were maleor female and smokers or non-smokers.

In one embodiment, the chemotherapeutic effect is potentiated in apatient suffering from adenocarcinoma who is treated with taxane andplatinum medicaments and is also given a medically sufficient dosage ofa Formula (I) compound so as to increase patient survival time in saidpatient suffering from adenocarcinoma.

In yet another embodiment, the chemotherapeutic effect is potentiated ina patient suffering from adenocarcinoma treated with paclitaxel, aFormula (I) compound, and cisplatin once every 2-4 weeks, wherein thedose of paclitaxel ranged from approximately 160 mg/m² to approximately190 mg/m², the dose of a Formula (I) compound ranged from approximately14 g/m² to approximately 22 g/m², and the dose of cisplatin ranged fromapproximately 60 mg/m² to approximately 100 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks was repeated at least once.

In still another embodiment, the chemotherapeutic effect is potentiatedin a patient suffering from adenocarcinoma treated with paclitaxel, aFormula (I) compound, and cisplatin once every 3 weeks, wherein the doseof paclitaxel was approximately 175 mg/m², the dose of a Formula (I)compound was approximately 18.4 g/m², and the dose of cisplatin rangedfrom approximately 75 mg/m² to approximately 85 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from adenocarcinoma weremale or female and smokers or non-smokers.

In one embodiment, hematological function is maintained or stimulated ina patient in need thereof, by providing to said patient a compositioncomprised of a Formula (I) compound in a medically sufficient dosage.

In one embodiment, a patient suffering from lung cancer treated withtaxane and/or platinum medicaments is given a medically sufficientdosage of a Formula (I) compound so as to maintain or stimulatehematological function in said patient suffering from lung cancer.

In another embodiment, the lung cancer is non-small cell lung carcinoma.

In yet another embodiment, the hematological function is maintained orstimulated in a patient suffering from lung cancer treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 2-4 weeks,wherein the dose of paclitaxel ranged from approximately 160 mg/m² toapproximately 190 mg/m², the dose of a Formula (I) compound ranged fromapproximately 14 g/m² to approximately 22 g/m², and the dose ofcisplatin ranged from approximately 60 mg/m² to approximately 100 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 2-4 weeks was repeated at least once.

In still another embodiment, the hematological function is maintained orstimulated in a patient suffering from lung cancer treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 3 weeks,wherein the dose of paclitaxel was approximately 175 mg/m², the dose ofa Formula (I) compound was approximately 18.4 g/m², and the dose ofcisplatin ranged from approximately 75 mg/m² to approximately 85 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from lung cancer were maleor female and smokers or non-smokers.

In one embodiment, the hematological function is maintained orstimulated in a patient suffering from adenocarcinoma who is treatedwith taxane and/or platinum medicaments and is also given a medicallysufficient dosage of a Formula (I) compound so as to maintain orstimulate hematological function in said patient suffering fromadenocarcinoma.

In yet another embodiment, the hematological function is maintained orstimulated in a patient suffering from adenocarcinoma treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 2-4 weeks,wherein the dose of paclitaxel ranged from approximately 160 mg/m² toapproximately 190 mg/m², the dose of a Formula (I) compound ranged fromapproximately 14 g/m² to approximately 22 g/m², and the dose ofcisplatin ranged from approximately 60 mg/m² to approximately 100 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 2-4 weeks was repeated at least once.

In still another embodiment, the hematological function is maintained orstimulated in a patient suffering from adenocarcinoma treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 3 weeks,wherein the dose of paclitaxel was approximately 175 mg/m², the dose ofa Formula (I) compound was approximately 18.4 g/m², and the dose ofcisplatin ranged from approximately 75 mg/m² to approximately 85 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from adenocarcinoma weremale or female and smokers or non-smokers.

In one embodiment, erythropoietin function or synthesis or homeostaticfunction of erythropoiesis is maintained or stimulated in a patient inneed thereof, by providing to said patient a composition comprised of aFormula (I) compound in a medically sufficient dosage.

In one embodiment, a patient suffering from lung cancer treated withtaxane and/or platinum medicaments is given a medically sufficientdosage of a Formula (I) compound so as to maintain or stimulateerythropoietin function or synthesis or homeostatic function oferythropoiesis in said patient suffering from lung cancer.

In another embodiment, the lung cancer is non-small cell lung carcinoma.

In yet another embodiment, the erythropoietin function or synthesis orhomeostatic function of erythropoiesis is maintained or stimulated in apatient suffering from lung cancer treated with paclitaxel, a Formula(I) compound, and cisplatin once every 2-4 weeks, wherein the dose ofpaclitaxel ranged from approximately 160 mg/m² to approximately 190mg/m², the dose of a Formula (I) compound ranged from approximately 14g/m² to approximately 22 g/m², and the dose of cisplatin ranged fromapproximately 60 mg/m² to approximately 100 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks was repeated at least once.

In still another embodiment, the erythropoietin function or synthesis orhomeostatic function of erythropoiesis is maintained or stimulated in apatient suffering from lung cancer treated with paclitaxel, a Formula(I) compound, and cisplatin once every 3 weeks, wherein the dose ofpaclitaxel was approximately 175 mg/m², the dose of a Formula (I)compound was approximately 18.4 g/m², and the dose of cisplatin rangedfrom approximately 75 mg/m² to approximately 85 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from lung cancer were maleor female and smokers or non-smokers.

In one embodiment, the erythropoietin function or synthesis orhomeostatic function of erythropoiesis is maintained or stimulated in apatient suffering from adenocarcinoma who is treated with taxane and/orplatinum medicaments and is also given a medically sufficient dosage ofa Formula (I) compound so as to maintain or stimulate erythropoietinfunction or synthesis or homeostatic function of erythropoiesis in saidpatient suffering from adenocarcinoma.

In yet another embodiment, the erythropoietin function or synthesis orhomeostatic function of erythropoiesis is maintained or stimulated in apatient suffering from adenocarcinoma treated with paclitaxel, a Formula(I) compound, and cisplatin once every 2-4 weeks, wherein the dose ofpaclitaxel ranged from approximately 160 mg/m² to approximately 190mg/m², the dose of a Formula (I) compound ranged from approximately 14g/m² to approximately 22 g/m², and the dose of cisplatin ranged fromapproximately 60 mg/m² to approximately 100 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks was repeated at least once.

In still another embodiment, the erythropoietin function or synthesis orhomeostatic function of erythropoiesis is maintained or stimulated in apatient suffering from adenocarcinoma treated with paclitaxel, a Formula(I) compound, and cisplatin once every 3 weeks, wherein the dose ofpaclitaxel was approximately 175 mg/m², the dose of a Formula (I)compound was approximately 18.4 g/m², and the dose of cisplatin rangedfrom approximately 75 mg/m² to approximately 85 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from adenocarcinoma weremale or female and smokers or non-smokers.

In one embodiment, anemia is mitigated or prevented in a patient in needthereof, by providing to said patient a composition comprised of aFormula (I) compound in a medically sufficient dosage.

In one embodiment, a patient suffering from lung cancer treated withtaxane and/or platinum medicaments is given a medically sufficientdosage of a Formula (I) compound so as to mitigate or preventchemotherapy-induced anemia in said patient suffering from lung cancer.

In another embodiment, the lung cancer is non-small cell lung carcinoma.

In yet another embodiment, chemotherapy-induced anemia is mitigated orprevented in a patient suffering from lung cancer treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 2-4 weeks,wherein the dose of paclitaxel ranged from approximately 160 mg/m² toapproximately 190 mg/m², the dose of a Formula (I) compound ranged fromapproximately 14 g/m² to approximately 22 g/m², and the dose ofcisplatin ranged from approximately 60 mg/m² to approximately 100 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 2-4 weeks was repeated at least once.

In still another embodiment, chemotherapy-induced anemia is mitigated orprevented in a patient suffering from lung cancer treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 3 weeks,wherein the dose of paclitaxel was approximately 175 mg/m², the dose ofa Formula (I) compound was approximately 18.4 g/m², and the dose ofcisplatin ranged from approximately 75 mg/m² to approximately 85 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from lung cancer were maleor female and smokers or non-smokers.

In one embodiment, chemotherapy-induced anemia is mitigated or preventedin a patient suffering from adenocarcinoma who is treated with taxaneand/or platinum medicaments and is also given a medically sufficientdosage of a Formula (I) compound so as to mitigate or preventchemotherapy-induced anemia.

In yet another embodiment, chemotherapy-induced anemia is mitigated orprevented in a patient suffering from adenocarcinoma treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 2-4 weeks,wherein the dose of paclitaxel ranged from approximately 160 mg/m² toapproximately 190 mg/m², the dose of a Formula (I) compound ranged fromapproximately 14 g/m² to approximately 22 g/m², and the dose ofcisplatin ranged from approximately 60 mg/m² to approximately 100 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 2-4 weeks was repeated at least once.

In still another embodiment, chemotherapy-induced anemia is mitigated orprevented in a patient suffering from adenocarcinoma treated withpaclitaxel, a Formula (I) compound, and cisplatin once every 3 weeks,wherein the dose of paclitaxel was approximately 175 mg/m², the dose ofa Formula (I) compound was approximately 18.4 g/m², and the dose ofcisplatin ranged from approximately 75 mg/m² to approximately 85 mg/m²,wherein said administration of paclitaxel, a Formula (I) compound, andcisplatin once every 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from adenocarcinoma weremale or female and smokers or non-smokers.

In one embodiment, pluripotent, multipotent, and unipotent normal stemcell function or synthesis is maintained or stimulated in a patient inneed thereof, by providing to said patient a composition comprised of aFormula (I) compound in a medically sufficient dosage.

In one embodiment, a patient suffering from lung cancer treated withtaxane and/or platinum medicaments is given a medically sufficientdosage of a Formula (I) compound so as to maintain or stimulatepluripotent, multipotent, and unipotent normal stem cell function orsynthesis in said patient suffering from lung cancer.

In another embodiment, the lung cancer is non-small cell lung carcinoma.

In yet another embodiment, pluripotent, multipotent, and unipotentnormal stem cell function or synthesis is maintained or stimulated in apatient suffering from lung cancer treated with paclitaxel, a Formula(I) compound, and cisplatin once every 2-4 weeks, wherein the dose ofpaclitaxel ranged from approximately 160 mg/m² to approximately 190mg/m², the dose of a Formula (I) compound ranged from approximately 14g/m² to approximately 22 g/m², and the dose of cisplatin ranged fromapproximately 60 mg/m² to approximately 100 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks was repeated at least once.

In still another embodiment, pluripotent, multipotent, and unipotentnormal stem cell function or synthesis is maintained or stimulated in apatient suffering from lung cancer treated with paclitaxel, a Formula(I) compound, and cisplatin once every 3 weeks, wherein the dose ofpaclitaxel was approximately 175 mg/m², the dose of a Formula (I)compound was approximately 18.4 g/m², and the dose of cisplatin rangedfrom approximately 75 mg/m² to approximately 85 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from lung cancer were maleor female and smokers or non-smokers.

In one embodiment, pluripotent, multipotent, and unipotent normal stemcell function or synthesis is maintained or stimulated in a patientsuffering from adenocarcinoma who is treated with taxane and/or platinummedicaments and is also given a medically sufficient dosage of a Formula(I) compound so as to maintain or stimulate pluripotent, multipotent,and unipotent normal stem cell function or synthesis in said patientsuffering from adenocarcinoma.

In yet another embodiment, pluripotent, multipotent, and unipotentnormal stem cell function or synthesis is maintained or stimulated in apatient suffering from adenocarcinoma treated with paclitaxel, a Formula(I) compound, and cisplatin once every 2-4 weeks, wherein the dose ofpaclitaxel ranged from approximately 160 mg/m² to approximately 190mg/m², the dose of a Formula (I) compound ranged from approximately 14g/m² to approximately 22 g/m², and the dose of cisplatin ranged fromapproximately 60 mg/m² to approximately 100 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 2-4 weeks was repeated at least once.

In still another embodiment, pluripotent, multipotent, and unipotentnormal stem cell function or synthesis is maintained or stimulated in apatient suffering from adenocarcinoma treated with paclitaxel, a Formula(I) compound, and cisplatin once every 3 weeks, wherein the dose ofpaclitaxel was approximately 175 mg/m², the dose of a Formula (I)compound was approximately 18.4 g/m², and the dose of cisplatin rangedfrom approximately 75 mg/m² to approximately 85 mg/m², wherein saidadministration of paclitaxel, a Formula (I) compound, and cisplatin onceevery 3 weeks was repeated for 6 cycles.

In another embodiment, the patients suffering from adenocarcinoma weremale or female and smokers or non-smokers.

In another embodiment, the Formula (I) compounds increase patientsurvival and/or delay tumor progression while maintaining or improvingthe quality of life of said patients diagnosed with lung cancer who arebeing treated with the taxane and/or platinum medicaments of the presentinvention.

In another embodiment, the lung cancer is non-small cell lung carcinoma.

In another embodiment, the Formula (I) compounds increase patientsurvival and/or delay tumor progression while maintaining or improvingthe quality of life of said patients diagnosed with adenocarcinoma whoare being treated with the taxane and/or platinum medicaments of thepresent invention.

In another embodiment, the patients suffering from adenocarcinoma weremale or female and smokers or non-smokers.

In another embodiment, the platinum medicaments of the present inventioninclude cisplatin, oxaliplatin, carboplatin, satraplatin, andderivatives and analogs thereof.

In another embodiment, the taxane medicament is selected from the groupconsisting of docetaxel, paclitaxel, paclitaxel derivatives,polyglutamylated forms of paclitaxel, liposomal paclitaxel, andderivatives and analogs thereof.

In still another embodiment, the compositions of Formula (I) include2,2′-dithio-bis-ethane sulfonate, a pharmaceutically-acceptable saltthereof, and/or an analog thereof, as well as prodrugs, analogs,conjugates, hydrates, solvates and polymorphs, as well as stereoisomers(including diastereoisomers and enantiomers) and tautomers of suchcompounds.

In still another embodiment, the dose rate of the taxane and platinummedicaments ranged from approximately 10-20 mg/m²/day and the dose rateof a Formula (I) compound ranged from approximately 4.1-41.0 g/m² perday; the concentration of the taxane and platinum medicaments and/orFormula (I) compounds is at least 0.01 mg/mL; the infusion time of thetaxane and platinum medicaments and/or Formula (I) compounds is fromapproximately 5 minutes to approximately 24 hours, and can be repeatedas needed and tolerated in a given patient; the schedule ofadministration of the taxane and platinum medicaments and/or Formula (I)compounds is every 2-8 weeks.

In another embodiment, a kit comprising a Formula (I) compound foradministration to a patient, and instructions for administering saidFormula (I) compound in an amount sufficient to cause one or more of thephysiological effects selected from the group consisting of increasingpatient survival time of said cancer patient receiving taxane andplatinum medicaments; causing a cytotoxic or apoptotic potentiation ofthe chemotherapeutic effects of said taxane and platinum medicaments;maintaining or stimulating hematological function in said patient,including said patient with cancer receiving chemotherapy; maintainingor stimulating erythropoietin function or synthesis in said patient,including said patient with cancer receiving chemotherapy; mitigating orpreventing anemia in said patient, including said patient with cancerreceiving chemotherapy; maintaining or stimulating pluripotent,multipotent, and unipotent normal stem cell function or synthesis insaid patient, including said patient with cancer receiving chemotherapy;promoting the arrest or retardation of tumor progression in said cancerpatient receiving taxane and/or platinum medicaments; and/or increasingpatient survival and/or delaying tumor progression while maintaining orimproving the quality of life in said cancer patient receiving taxaneand platinum medicaments.

In another embodiment, the cancer patient has lung cancer.

In yet another embodiment, the lung cancer is non-small cell lungcancer.

In still another embodiment, the cancer patient has an adenocarcinoma.

In one embodiment, the kit further contains instructions foradministering a taxane medicament and a platinum medicament selectedfrom the group consisting of cisplatin, oxaliplatin, carboplatin,satraplatin, and derivatives and analogs thereof.

In another embodiment, the kit further contains instructions foradministering a platinum medicament and a taxane medicament selectedfrom the group consisting of docetaxel, paclitaxel, polyglutamylatedforms of paclitaxel, liposomal paclitaxel, and derivatives and analogsthereof.

In yet another embodiment, the platinum and taxane medicaments arecisplatin and paclitaxel.

Chemotherapeutic agents may be prepared and administered to subjectsusing methods known within the art. For example, paclitaxel may beprepared using methods described in U.S. Pat. Nos. 5,641,803, 6,506,405,and 6,753,006 and is administered as known in the art (see, e.g., U.S.Pat. Nos. 5,641,803, 6,506,405, and 6,753,006). Paclitaxel may beprepared for administration in a dose in the range of about 50 mg/m² toabout 275 mg/m². Preferred doses include about 160 mg/m² to about 190mg/m². The most preferred dose is about 175 mg/m².

Docetaxel may be prepared using methods described in U.S. Pat. No.4,814,470 and is administered as known in the art (see, e.g., U.S. Pat.Nos., 4,814,470, 5,438,072, 5,698,582, and 5,714,512). Docetaxel may beprepared for administration in a dose in the range of about 30 mg/m² toabout 100 mg/m². Preferred doses include about 55 mg/m², about 60 mg/m²,about 75 mg/m², and about 100 mg/m².

Cisplatin may be prepared using methods described in U.S. Pat. Nos.4,302,446, 4,322,391, 4,310,515, and 4,915,956 and is administered asknown in the art (see, e.g., U.S. Pat. Nos. 4,177,263, 4,310,515,4,451,447). Cisplatin may be prepared for administration in a dose inthe range of about 30 mg/m² to about 120 mg/m² in a single dose.Preferred doses range from about 60 mg/m² to about 100 mg/m². The mostpreferred doses range from about 75 mg/m² to about 85 mg/m².

Carboplatin may be prepared using methods described in U.S. Pat. No.4,657,927 and is administered as known in the art (see, e.g., U.S. Pat.No. 4,657,927). Carboplatin may be prepared for administration in a dosein the range of about 20 mg/kg and about 200 mg/kg. Preferred dosesinclude about 300 mg/m² and about 360 mg/m². Other dosing may becalculated using a formula according to the manufacturer's instructions.

Oxaliplatin may be prepared using methods described in U.S. Pat. Nos.5,290,961, 5,420,319, 5,338,874 and is administered as known in the art(see, e.g., U.S. Pat. No. 5,716,988). Oxaliplatin may be prepared foradministration in a dose in the range of about 50 mg/m² and about 200mg/m². Preferred doses include about 85 mg/m² and about 130 mg/m².

The compositions of Formula (I) include 2,2′-dithio-bis-ethanesulfonate, a pharmaceutically-acceptable salt thereof, and/or an analogthereof, as well as prodrugs, analogs, conjugates, hydrates, solvatesand polymorphs, as well as stereoisomers (including diastereoisomers andenantiomers) and tautomers of such compounds.Pharmaceutically-acceptable salts of the present invention include, butare not limited to: (i) a monosodium salt; (ii) a sodium potassium salt;(iii) a dipotassium salt; (iv) a calcium salt; (v) a magnesium salt;(vi) a manganese salt; (vii) an ammonium salt; (viii) a monopotassiumsalt; and (ix) most preferably, disodium. It should be noted that mono-and di-potassium salts are only administered to a subject if the totaldose of potassium administered at any given point in time is not greaterthan 100 Meq., the subject is not hyperkalemic, and/or the subject doesnot have a condition that would predispose the subject to hyperkalemia(e.g., renal failure).

By way of non-limiting example, disodium 2,2′-dithio-bis-ethanesulfonate (also referred to in the literature as dimesna, Tavocept™, andBNP7787) is a known compound and can be manufactured by methods known inthe art. See, e.g., J. Org. Chem. 26:1330-1331 (1961); J. Org. Chem.59:8239 (1994). In addition, various salts of 2,2′-dithio-bis-ethanesulfonate, as well as other dithioethers may also be synthesized asoutlined in U.S. Pat. No. 5,808,160, U.S. Pat. No. 6,160,167 and U.S.Pat. No. 6,504,049. Compounds of Formula (I) may be manufactured asdescribed in Published U.S. Patent Application 2005/0256055. Thedisclosures of these patents, patent applications, and published patentapplications are incorporated herein by reference, in their entirety.

Preferred doses of the Formula (I) compounds of the present inventionrange from about 14 g/m² to about 22 g/m², with a most preferred dose of18.4 g/m².

In certain of the methods of the invention, as well as in the uses ofthe compositions and formulations of the invention, the Formula (I)compound may be administered in conjunction with one or morechemotherapeutic agent, wherein each course being of a specified perioddependent upon the specific chemotherapeutic agent or agents utilized.In conjunction with the inventions described and claimed herein, thetreatment regimens may be comprised, for example, of two or moretreatment courses, of five or more treatment courses, of six or moretreatment courses, of seven or more treatment courses, of eight or moretreatment courses, or of nine or more treatment courses. The treatmentcourses may also be continuous in nature.

The compositions and formulations of the present invention, alone or incombination with one or more chemotherapeutic agents, and instructionsfor their use, may be included in a form of packs or kits. Thus, theinvention also includes kits comprising the compositions, formulations,and/or devices described herein with instructions for use. For example,a kit may comprise a Formula (I) compound of the present invention andinstructions for administration. Kits may additionally comprise one ormore chemotherapeutic agents with instructions for their use. Kits mayalso additionally comprise one or more pre-treatments as describedherein and instructions for their use.

Aspects of the present invention also include controlled delivery orother doses, dosage forms, formulations, compositions and/or devicescontaining a Formula (I) compound of the present invention, whichinclude, e.g., 2,2′-dithio-bis-ethane sulfonate, apharmaceutically-acceptable salt or an analog thereof; or a mesnaheteroconjugate; as well as one or more chemotherapeutic agents. Thesecompositions are comprised of, for example, various doses and dosageforms for: (i) oral (e.g., tablet, suspension, solution, gelatin capsule(hard or soft), sublingual, dissolvable tablet, troche, and the like),or with sublingual administration which avoids first-pass metabolismthrough the liver (i.e., the cytochrome P₄₅₀ oxidase system); (ii)injection (e.g., subcutaneous administration, intradermaladministration, subdermal administration, intramuscular administration,depot administration, intravenous administration, intra-arterialadministration, and the like), wherein the administration may occur by,e.g., injection delivery, delivery via parenteral bolus, slowintravenous injection, and intravenous drip, and infusion devices (e.g.,implantable infusion devices, both active and passive); (iii)intra-cavitary (e.g., into the intrapleural, intraperitoneal,intravesicular, and/or intrathecal spaces); (iv) per rectum (e.g.,suppository, retention enema); and (v) topical administration routes tosubjects as treatment for various cancers.

Examples of dosage forms suitable for injection of the compounds andformulations of the present invention include delivery via bolus such assingle or multiple or continuous or constant administrations byintravenous injection, subcutaneous, subdermal, and intramuscularadministration. These forms may be injected using syringes, pens, jetinjectors, and internal or external pumps, with vascular or peritonealaccess, for example. Syringes come in a variety sizes including 0.3,0.5, 1, 2, 5, 10, 25 and 50 mL capacity. Needleless jet injectors arealso known in the art and use a pressurized air to inject a fine sprayof solution into the skin. Pumps are also known in the art. The pumpsare connected by flexible tubing to a catheter, which is inserted intothe tissue just below the skin. The catheter is left in place forseveral days at a time. The pump is programmed to dispense the necessaryamount of solution at the proper times.

Examples of infusion devices for compounds and formulations of thepresent invention include infusion pumps containing a Formula (I)compound of the present invention to be administered at a desired rateand amount for a desired number of doses or steady state administration,and include implantable drug pumps.

Examples of implantable infusion devices for compounds and formulationsof the invention include any solid form or liquid form in which theactive agent is a solution, suspension or encapsulated within ordispersed throughout a biodegradable polymer or synthetic polymer, forexample, silicone, polypropylene, silicone rubber, silastic or similarpolymer.

Examples of controlled release drug formulations useful for delivery ofthe compounds and formulations of the invention are found in, forexample, Sweetman, S. C. (Ed.)., The Complete Drug Reference, 33rdEdition, Pharmaceutical Press, Chicago, 2483 pp. (2002); Aulton, M. E.(Ed.), Pharmaceutics: The Science of Dosage Form Design. ChurchillLivingstone, Edinburgh, 734 pp. (2000); and, Ansel, H. C., Allen, L. V.and Popovich, N. G., Pharmaceutical Dosage Forms and Drug DeliverySystems, 7th Ed., Lippincott, 676 pp. (1999). Excipients employed in themanufacture of drug delivery systems are described in variouspublications known to those skilled in the art including, for example,Kibbe, E. H., Handbook of Pharmaceutical Excipients, 3rd Ed., AmericanPharmaceutical Association, Washington, 665 pp. (2000).

Further examples of dosage forms of the present invention primarilyutilized with oral administration, include but are not limited to,modified-release (MR) dosage forms including delayed-release (DR) forms;prolonged-action (PA) forms; controlled-release (CR) forms;extended-release (ER) forms; timed-release (TR) forms; and long-acting(LA) forms. As previously stated, these formulations are often used withorally administered dosage forms, however these terms may be applicableto any of the dosage forms, formulations, compositions and/or devicesdescribed herein. These formulations delay and control total drugrelease for some time after drug administration, and/or drug release insmall aliquots intermittently after administration, and/or drug releaseslowly at a controlled rate governed by the delivery system, and/or drugrelease at a constant rate that does not vary, and/or drug release for asignificantly longer period than usual formulations.

Modified-release dosage forms of the present invention include dosageforms having drug release features based on time, course, and/orlocation which are designed to accomplish therapeutic or convenienceobjectives not offered by conventional or immediate-release forms. See,e.g., Bogner, R. H., Bioavailability and bioequivalence ofextended-release oral dosage forms. U.S. Pharmacist 22:3-12 (1997).Extended-release dosage forms of the invention include, for example, asdefined by the FDA, a dosage form that allows a reduction in dosingfrequency to that represented by a conventional dosage form, e.g., asolution or an immediate-release dosage form.

For example, one embodiment provides extended-release formulationscontaining a Formula (I) compound of the present invention forparenteral administration. Extended rates of activity of a Formula (I)compound of the present invention following injection may be achieved ina number of ways, including the following: crystal or amorphous Formula(I) compound forms having prolonged dissolution characteristics; slowlydissolving chemical complexes of Formula (I) compound formulations;solutions or suspensions of a Formula (I) compound of the presentinvention in slowly absorbed carriers or vehicles (e.g., oleaginous);increased particle size of a Formula (I) compound of the presentinvention, in suspension; or, by injection of slowly erodingmicrospheres of said Formula (I) compounds (see, e.g., Friess, W., etal., Insoluble collagen matrices for prolonged delivery of proteins.Pharmaceut. Dev. Technol. 1:185-193 (1996)). For example, the durationof action of the various forms of insulin is based in part on itsphysical form (i.e., amorphous or crystalline), complex formation withadded agents, and its dosage form (i.e., solution or suspension).

An acetate, phosphate, citrate, bicarbonate, glutamine or glutamatebuffer may be added to modify pH of the final composition. Optionally acarbohydrate or polyhydric alcohol tonicifier and, a preservativeselected from the group consisting of m-cresol, benzyl alcohol, methyl,ethyl, propyl and butyl parabens and phenol may also be added. Water forinjection, tonicifying agents such as sodium chloride, as well as otherexcipients, may also be present, if desired. For parenteraladministration, formulations may be isotonic or substantially isotonicto avoid irritation and pain at the site of administration.Alternatively, formulations for parenteral administration may also behyperosmotic relative to normal mammalian plasma, as described herein.

The terms buffer, buffer solution and buffered solution, when used withreference to hydrogen-ion concentration or pH, refer to the ability of asolute/solvent system, particularly an aqueous solution, to resist achange in pH with the addition of acid or alkali, or upon dilution witha solvent, or both. Characteristic of buffered solutions, which undergosmall changes of pH on addition of acid or base, is the presence eitherof a weak acid and a salt of the weak acid, or a weak base and a salt ofthe weak base. An example of the former system is acetic acid and sodiumacetate. The change of pH is slight as long as the amount of hydroxylion added does not exceed the capacity of the buffer system toneutralize it. The buffer used in the practice of the present inventionis selected from any of the following, for example, an acetate,phosphate, citrate, bicarbonate, glutamine, or glutamate buffer, withthe most preferred buffer being a phosphate buffer.

Carriers or excipients can also be used to facilitate administration ofthe compositions and formulations of the invention. Examples of carriersand excipients include calcium carbonate, calcium phosphate, varioussugars such as lactose, glucose, or sucrose, or types of starch,cellulose derivatives, gelatin, polyethylene glycols, andphysiologically compatible solvents.

A stabilizer may be included in the formulations of the invention, butwill generally not be needed. If included, however, a stabilizer usefulin the practice of the invention is a carbohydrate or a polyhydricalcohol. The polyhydric alcohols include such compounds as sorbitol,mannitol, glycerol, xylitol, and polypropylene/ethylene glycolcopolymer, as well as various polyethylene glycols (PEG) of molecularweight 200, 400, 1450, 3350, 4000, 6000, and 8000). The carbohydratesinclude, for example, mannose, ribose, trehalose, maltose, inositol,lactose, galactose, arabinose, or lactose.

The United States Pharmacopeia (USP) states that anti-microbial agentsin bacteriostatic or fungistatic concentrations must be added topreparations contained in multiple dose containers. They must be presentin adequate concentration at the time of use to prevent themultiplication of microorganisms inadvertently introduced into thepreparation while withdrawing a portion of the contents with ahypodermic needle and syringe, or using other invasive means fordelivery, such as pen injectors. Antimicrobial agents should beevaluated to ensure compatibility with all other components of theformulation, and their activity should be evaluated in the totalformulation to ensure that a particular agent that is effective in oneformulation is not ineffective in another. It is not uncommon to findthat a particular agent will be effective in one formulation but noteffective in another formulation.

A preservative is, in the common pharmaceutical sense, a substance thatprevents or inhibits microbial growth and may be added to apharmaceutical formulation for this purpose to avoid consequent spoilageof the formulation by microorganisms. While the amount of thepreservative is not great, it may nevertheless affect the overallstability of the Formula (I) compound of the present invention.Preservatives include, for example, benzyl alcohol and ethyl alcohol.While the preservative for use in the practice of the invention canrange from 0.005 to 1.0% (w/v), the preferred range for eachpreservative, alone or in combination with others, is: benzyl alcohol(0.1-1.0%), or m-cresol (0.1-0.6%), or phenol (0.1-0.8%) or combinationof methyl (0.05-0.25%) and ethyl or propyl or butyl (0.005%-0.03%)parabens. The parabens are lower alkyl esters of para-hydroxybenzoicacid. A detailed description of each preservative is set forth in“Remington's Pharmaceutical Sciences” as well as Pharmaceutical DosageForms: Parenteral Medications, Vol. 1, Avis, et al. (1992). For thesepurposes, the 2,2′-dithio-bis-ethane sulfonate, apharmaceutically-acceptable salt thereof, an analog thereof, and/or acompound of Formula (I), may be administered parenterally (includingsubcutaneous injections, intravenous, intramuscular, intradermalinjection or infusion techniques) in dosage unit formulations containingconventional non-toxic pharmaceutically acceptable carriers, adjuvants,and vehicles. In addition, formulations of the present inventiondesigned for parenteral administration must be stable, sterile,pyrogen-free, and possess particulate levels and size within acceptedlevels.

If desired, the parenteral formulation may be thickened with athickening agent such as a methylcellulose. The formulation may beprepared in an emulsified form, either water in oil or oil in water. Anyof a wide variety of pharmaceutically-acceptable emulsifying agents maybe employed including, for example, acacia powder, a non-ionicsurfactant, or an ionic surfactant.

It may also be desirable to add suitable dispersing or suspending agentsto the pharmaceutical formulation. These may include, for example,aqueous suspensions such as synthetic and natural gums, e.g.,tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose,methylcellulose, polyvinyl-pyrrolidone, or gelatin.

It is possible that other ingredients may be present in the parenteralpharmaceutical formulation of the invention. Such additional ingredientsmay include wetting agents, oils (e.g., a vegetable oil such as sesame,peanut, or olive), analgesic agents, emulsifiers, antioxidants, bulkingagents, tonicity modifiers, metal ions, oleaginous vehicles, proteins(e.g., human serum albumin, gelatin, or proteins) and a zwitterion(e.g., an amino acid such as betaine, taurine, arginine, glycine,lysine, or histidine). Such additional ingredients, of course, shouldnot adversely affect the overall stability of the pharmaceuticalformulation of the present invention.

Containers and kits are also a part of a composition and may beconsidered a component. Therefore, the selection of a container is basedon a consideration of the composition of the container, as well as ofthe ingredients, and the treatment to which it will be subjected.

Suitable routes of parenteral administration include intramuscular,intravenous, subcutaneous, intraperitoneal, subdermal, intradermal,intraarticular, intrathecal, and the like. Mucosal delivery is alsopermissible. The dose and dosage regimen will depend upon the weight,health, disease type, and degree of disease severity within the subject.Regarding pharmaceutical formulations, see, Pharmaceutical Dosage Forms:Parenteral Medications, Vol. 1, 2nd ed., Avis et al., Eds., MarcelDekker, New York, N.Y. (1992).

In addition to the above means of achieving extended drug action, therate and duration of delivery of a Formula (I) compound of the presentinvention, as well as one or more chemotherapeutic agents may becontrolled by, e.g., using mechanically controlled drug infusion pumps.

The present invention, in part, provides infusion dose deliveryformulations and devices, including but not limited to, implantableinfusion devices for delivery of compositions and formulations of theinvention. Implantable infusion devices may employ inert material suchas the biodegradable polymers described above or synthetic silicones,for example, cylastic, silicone rubber or other commercially-availablepolymers manufactured and approved for such uses. The polymer may beloaded with a Formula (I) compound of the present invention and anyexcipients. Implantable infusion devices may also comprise the coatingof, or a portion of, a medical device wherein the coating comprises thepolymer loaded with a Formula (I) compound of the present invention, oneor more chemotherapeutic agents, and any excipient. Such an implantableinfusion device may be prepared as disclosed in U.S. Pat. No. 6,309,380by coating the device with an in vivo biocompatible and biodegradable orbioabsorbable or bioerodable liquid or gel solution containing a polymerwith the solution comprising a desired dosage amount of a Formula (I)compound of the present invention, one or more chemotherapeutic agents,and any excipients. The solution is converted to a film adhering to themedical device thereby forming the implantable Formula (I)compound-deliverable medical device.

An implantable infusion device may also be prepared by the in situformation of a Formula (I) compound of the present invention, containinga solid matrix (as disclosed in U.S. Pat. No. 6,120,789, the disclosureof which is hereby incorporated by reference, in its entirety) and oneor more chemotherapeutic agents. Implantable infusion devices may bepassive or active. An active implantable infusion device may comprise aFormula (I) compound reservoir, a means of allowing the Formula (I)compound to exit the reservoir, for example a permeable membrane, and adriving force to propel the Formula (I) compound from the reservoir. Thereservoir of the aforementioned active implantable infusion device mayalso contain one or more chemotherapeutic agents. Such an activeimplantable infusion device may additionally be activated by anextrinsic signal, such as that disclosed in WO 02/45779, wherein theimplantable infusion device comprises a system configured to deliver aFormula (I) compound of the present invention and one or morechemotherapeutic agents, comprising an external activation unit operableby a user to request activation of the implantable infusion device,including a controller to reject such a request prior to the expirationof a lockout interval. Examples of an active implantable infusion deviceinclude implantable drug pumps. Implantable drug pumps include, forexample, miniature, computerized, programmable, refillable drug deliverysystems with an attached catheter that inserts into a target organsystem, usually the spinal cord or a vessel. See, Medtronic Inc.Publications: UC9603124EN NP-2687, 1997; UC199503941b EN NP-2347182577-101, 2000; UC199801017a EN NP3273a 182600-101, 2000; UC200002512EN NP4050, 2000; UC199900546bEN NP-3678EN, 2000. Medtronic, Inc.,Minneapolis, Minn. (1997-2000). Many pumps have 2 ports: one into whichdrugs can be injected and the other that is connected directly to thecatheter for bolus administration or analysis of fluid from thecatheter. Implantable drug infusion pumps (e.g., SynchroMed EL andSynchroMed programmable pumps; Medtronic) are indicated for long-termintrathecal infusion of sulfate for the treatment of chronic intractablepain; intravascular infusion of floxuridine for treatment of primary ormetastatic cancer; intrathecal injection (baclofen injection) for severespasticity; long-term epidural infusion of morphine sulfate fortreatment of chronic intractable pain; long-term intravascular infusionof doxorubicin, cisplatin, or methotrexate for the treatment ormetastatic cancer; and long-term intravenous infusion of clindamycin forthe treatment of osteomyelitis. Such pumps may also be used for thelong-term infusion of one or more compounds simultaneously, including, aFormula (I) compound of the present invention, in combination with oneor more chemotherapeutic agents of choice, at a desired amount for adesired number of doses or steady state administration. One form of atypical implantable drug infusion pump (e.g., SynchroMed EL programmablepump; Medtronic) is titanium covered and roughly disk shaped, measures85.2 mm in diameter and 22.86 mm in thickness, weighs 185 g, has a drugreservoir of 10 mL, and runs on a lithium thionyl-chloride battery witha 6- to 7-year life, depending on use. The downloadable memory containsprogrammed drug delivery parameters and calculated amount of drugremaining, which can be compared with actual amount of drug remaining toaccess accuracy of pump function, but actual pump function over time isnot recorded. The pump is usually implanted in the right or leftabdominal wall. Other pumps useful in the present invention include, forexample, Portable Disposable Infuser Pumps (PDIPs). Additionally,implantable infusion devices may employ liposome delivery systems, suchas a small unilamellar vesicles, large unilamellar vesicles, andmultilamellar vesicles that can be formed from a variety ofphospholipids, such as cholesterol, stearyl amine, orphosphatidylcholines.

The present invention also provides in part dose delivery formulationsand devices formulated to enhance bioavailability of a Formula (I)compound of the present invention. This may be in addition to, or incombination with, one or more chemotherapeutic agents, or any of theformulations and/or devices described above.

For example, an increase in bioavailability of a Formula (I) compound ofthe present invention, may be achieved by complexation of a Formula (I)compound with one or more bioavailability or absorption enhancing agentsor formulations, including bile acids such as taurocholic acid.

The present invention also provides for the formulation of an oxidativemetabolism-affecting Formula (I) compound of the present invention, aswell as one or more chemotherapeutic agents, in a microemulsion toenhance bioavailability. A microemulsion is a fluid and stablehomogeneous solution composed of four major constituents, respectively,a hydrophilic phase, a lipophilic phase, at least one surfactant (SA)and at least one cosurfactant (CoSA). A surfactant is a chemicalcompound possessing two groups, the first polar or ionic, which has agreat affinity for water, the second which contains a longer or shorteraliphatic chain and is hydrophobic. These chemical compounds havingmarked hydrophilic character are intended to cause the formation ofmicelles in aqueous or oily solution. Examples of suitable surfactantsinclude mono-, di- and triglycerides and polyethylene glycol (PEG) mono-and diesters. A cosurfactant, also sometimes known as “co-surface-activeagent”, is a chemical compound having hydrophobic character, intended tocause the mutual solubilization of the aqueous and oily phases in amicroemulsion. Examples of suitable co-surfactants include ethyldiglycol, lauric esters of propylene glycol, oleic esters ofpolyglycerol, and related compounds.

Any such dose may be administered by any of the routes or in any of theforms herein described. For example, a dose or doses could be givenparenterally using a dosage form suitable for parenteral administrationwhich may incorporate features or compositions described in respect ofdosage forms delivered in a modified release, extended release, delayedrelease, slow release or repeat action oral dosage form.

The present invention also provides for the formulation of an oxidativemetabolism-affecting Formula (I) compound of the present invention, forrectal delivery and absorption via the utilization of rectalsuppositories or retention enemas. Generally, suppositories are utilizedfor delivery of drugs to the rectum and sigmoid colon. The idealsuppository base for the delivery of the formulations of the presentinvention should meet the following specifications: (i) a base which isnon-toxic and non-irritating to the anal mucous membranes; (ii) a basewhich is compatible with a variety of drugs; (iii) a base which melts ordissolves in rectal fluids; and (iv) a base which is stable in storageand does not bind or otherwise interfere with the release and/orabsorption of the pharmaceutical formulations contained therein. Typicalsuppository bases include: cocoa butter, glycerinated gelatine,hydrogenated vegetable oils, mixtures of polyethylene glycols of variousmolecular weights and fatty acid esters of polyethylene glycol. Therectal Epithelium is lipoidal in character. The lower, middle, and upperhemorrhoidal veins surrounds the rectum. Only the upper vein conveysblood into the portal system, thus drugs absorbed into the lower andmiddle hemorrhoidal veins will bypass the liver and the cytochrome P₄₅₀oxidase system. Absorption and distribution of a drug is thereforemodified by its position within the rectum, in that at least a portionof the drug absorbed from the rectum may pass directly into the inferiorvena cava, bypassing the liver. The present invention also provides forthe formulation of a Formula (I) compound of the present invention, aswell as one or more chemotherapeutic agents, administered bysuppository.

Various representative Formula (I) compounds of the present inventionhave been synthesized and purified. Additionally, disodium2,2′-dithio-bis ethane sulfonate (also referred to in the literature asTavocept™, dimesna, and BNP7787), has been introduced into Phase I,Phase II, and Phase III clinical testing in patients, as well as innon-clinical testing, by the Assignee, BioNumerik Pharmaceuticals, Inc.,with guidance provided by the Applicant of the instant invention and ina U.S. Phase II NSCLC Clinical Trial, whose resulting data was furtheranalyzed by the Assignee, BioNumerik Pharmaceuticals, Inc., again withguidance provided by the Applicant of the instant invention. Forexample, the data from the Jpan Phase III Clinical Trial and the U.S.Phae II Clinical Trial utilizing disodium 2,2′-dithio-bis ethanesulfonate (Tavocept™ with one or more chemotherapeutic agents havedemonstrated the ability of disodium 2,2′-dithio-bis ethane sulfonate tomarkedly increase the survival time of individuals with non-small celllung carcinoma (NSCLC), including adenocarcinoma. In brief, experimentalevidence supports the finding that disodium 2,2′-dithio-bis ethanesulfonate functions to increase patient survival time by increasingoxidative metabolism within tumor cells in a selective manner. Moreover,these clinical results have also demonstrated the ability of disodium2,2′-dithio-bis ethane sulfonate to reduce both the frequency andseverity of deleterious chemotherapeutic agent-induced physiologicalside effects and pharmacological effects on normal (i.e., non-cancerous)cells and tissues, while concomitantly avoiding any diminution of thecytotoxic effect of the chemotherapeutic agent in cancer cells.

V. Pharmacology of Taxanes

Taxanes are semi-synthetically derived analogues of naturally occurringcompounds derived from plants. In particular, taxanes are derived fromthe needles and twigs of the European yew (Taxus baccata), or the barkof the Pacific yew (Taxus brevifolia). The most widely known taxanes atthis time are paclitaxel (Taxol®) and docetaxel (Taxotere®), which arewidely distributed as antineoplastic agents.

Paclitaxel was discovered in the late 1970s, and was found to be aneffective antineoplastic agent with a mechanism of action different fromthen-existing chemotherapeutic agents. Taxanes are recognized aseffective agents in the treatment of many solid tumors which arerefractory to other antineoplastic agents.

Paclitaxel has the molecular structure shown below as Formula (A):

Docetaxel is an analog of Paclitaxel, and has the molecular structureshown below as Formula (B):

Taxanes exert their biological effects on the cell microtubules and actto promote the polymerization of tubulin, a protein subunit of spindlemicrotubules. The end result is the inhibition of depolymerization ofthe microtubules, which causes the formation of stable and nonfunctionalmicrotubules. This disrupts the dynamic equilibrium within themicrotubule system, and arrests the cell cycle in the late G₂ and Mphases, which inhibits cell replication. Taxanes interfere with thenormal function of microtubule growth and arrests the function ofmicrotubules by hyper-stabilizes their structure. This destroys thecell's ability to use its cytoskeleton in a flexible manner.

Taxanes function as an anti-neoplastic agent by binding to theN-terminal 31 amino acid residues of the β-tubulin subunit in tubulinoligomers or polymers, rather than tubulin dimers. Unlike otheranti-microtubule agents (e.g., vinca alkaloids) which preventmicrotubule assembly, submicromolar concentrations of taxanes functionto decrease the lag-time and shift the dynamic equilibrium betweentubulin dimers and microtubules (i.e., the hyperpolymerization oftubulin oligomers) toward microtubules assembly and stabilize the newlyformed microtubules against depolymerization. The microtubules which areformed are highly stable, thereby inhibiting the dynamic reorganizationof the microtubule network. See, e.g., Rowinsky, E. K., et al., Taxol:The prototypic taxane, an important new class of antitumor agents.Semin. Oncol. 19:646 (1992). Tubulin is the “building block” ofmicrotubules, the resulting microtubule/taxane complex does not have theability to disassemble. Thus, the binding of taxanes inhibit the dynamicreorganization of the microtubule network. This adversely affects cellfunction because the shortening and lengthening of microtubules (i.e.,dynamic instability) is necessary for their function as a mechanism totransport other cellular components. For example, during mitosis,microtubules position the chromosomes during their replication andsubsequent separation into the two daughter-cell nuclei.

In addition, even at submicromolar concentrations, the taxanes alsoinduce microtubule bundling in cells, as well as the formation ofnumerous abnormal mitotic asters (which unlike mitotic asters formedunder normal physiological conditions, do not require centrioles forenucleation. Thus, the taxanes function to inhibit the proliferation ofcells by inducing a sustained mitotic “block” at the metaphase-anaphaseboundary at a much lower concentration than that required to increasemicrotubule polymer mass and microtubule bundle formation. See, e.g.,Rao, S., et al., Direct photoaffinity labeling of tubulin with taxol. J.Natl. Cancer Inst. 84:785 (1992). It should be noted that many of thedeleterious physiological side-effects caused by the taxanes are causedby the sustained mitotic “block” at the metaphase-anaphase boundary innormal (i.e., non-neoplastic cells).

In addition to stabilizing microtubules, the taxane, paclitaxel, may actas a “molecular sponge” by sequestering free tubulin, thus effectivelydepleting the cells supply of tubulin monomers and/or dimers. Thisactivity may trigger the aforementioned apoptosis. One commoncharacteristic of most cancer cells is their rapid rate of celldivision. In order to accommodate this, the cytoskeleton of the cancercell undergoes extensive restructuring. Paclitaxel is an effectivetreatment for aggressive cancers because it adversely affects theprocess of cell division by preventing this restructuring. Althoughnon-cancerous cells are also adversely affected, the rapid division rateof cancer cells make them far more susceptible to paclitaxel treatment.

Further research has also indicated that paclitaxel, induces programmedcell death (apoptosis) in cancer cells by binding to an apoptosisstopping protein called B-cell leukemia 2 (Bcl-2), thus arresting itsfunction.

The molecular structure of the taxanes are complex alkaloid estersconsisting of a taxane system linked to a four-member oxetan ring atpositions C-4 and C-5. The taxane rings of both paclitaxel anddocetaxel, but not 10-deacetylbaccatin III, are linked to an ester atthe C-13 position. Experimental and clinical studies have demonstratedthat analogs lacking the aforementioned linkage have very littleactivity against mammalian tubulin. Moreover, the moieties at C-2′ andC-3′ are critical with respect to its full biological activity,specifically, for the anti-microtubule hyperpolymerization effect oftaxane. The C-2′ —OH is of paramount importance for the activity oftaxol and the Formula (I) compounds of the present invention, and whilethe C-2′ —OH of taxol can be “substituted” by a sufficiently strongnucleophile (see, PCT/US98/21814; page 62, line 8-27) the biologicalactivity would be greatly diminished. See, e.g., Lataste, H., et al.,Relationship between the structures of Taxol and baccatine IIIderivatives and their in vitro action of the disassembly of mammalianbrain. Proc. Natl. Acad. Sri. 81.4090 (1984). For example, it has beendemonstrated that the substitution of an acetyl group at the C-2′position markedly reduces taxane activity. See, e.g.,Gueritte-Voegelein, F., et al., Relationships between the structures oftaxol analogues and their antimitotic activity. J. Med. Chem. 34:992(1991).

Taxanes are toxic compounds having a low therapeutic index which havebeen shown to cause a number of different toxic effects in patients. Themost well-known and severe adverse effects of taxanes are neurotoxicityand hematologic toxicity, particularly anemia and severeneutropenia/thrombocytopenia. Additionally, taxanes also causehypersensitivity reactions in a large percentage of patients;gastrointestinal effects (e.g., nausea, diarrhea and vomiting);alopecia; anemia; and various other deleterious physiological effects,even at the recommended dosages. The Taxane medicaments disclosed in thepresent invention include, in a non-limiting manner, docetaxel orpaclitaxel (including the commercially-available paclitaxel derivativesTaxol® and Abraxane®), polyglutamylated forms of paclitaxel (e.g.,Xyotax®), liposomal paclitaxel (e.g., Tocosol®), and analogs andderivatives thereof.

VI. Pharmacology of Platinum Compounds

The anti-neoplastic drug cisplatin (cis-diamminedichloroplatinum or“CDDP”), and related platinum based drugs including carboplatin andoxaliplatin, are widely used in the treatment of a variety ofmalignancies including, but not limited to, cancers of the ovary, lung,colon, bladder, germ cell tumors and head and neck. Platinum agents arereported to act, in part, by aquation (i.e., to form reactive aquaspecies), some of which may predominate intracellularly, andsubsequently form DNA intra-strand coordination chelation cross-linkswith purine bases, thereby cross-linking DNA. The currently acceptedparadigm with respect to cisplatin's mechanism of action is that thedrug induces its cytotoxic properties by forming a reactive monoaquospecies that reacts with the N⁷ nitrogen contained within the imidazolecomponents of guanine and adenosine found in nuclear DNA to formintrastrand platinum-DNA adducts. However, the exact mechanism of actionof cisplatin is not completely understood and remains a subject ofresearch interest within the scientific community. Thus, this mechanismis believed to work predominantly through intra-strand cross-links, andless commonly, through inter-strand cross-links, thereby disrupting theDNA structure and function, which is cytotoxic to cancer cells.Platinum-resistant cancer cells are resilient to the cytotoxic actionsof these agents. Certain cancers exhibit intrinsic de novo naturalresistance to the killing effects of platinum agents and undergo noapoptosis, necrosis or regression following initial platinum compoundtreatment. In contrast, other types of cancers exhibit cytotoxicsensitivity to platinum drugs, as evidenced by tumor regressionfollowing initial treatment, but subsequently develop an increasinglevel of platinum resistance, which is manifested as a reducedresponsiveness and/or tumor growth following treatment with the platinumdrug (i.e., “acquired resistance”). Accordingly, new platinum agents arecontinually being sought which will effectively kill tumor cells, butthat are also insensitive or less susceptible to tumor-mediated drugresistance mechanisms that are observed with other platinum agents.

The reaction for cisplatin hydrolysis is illustrated below in Scheme I:

In neutral pH (i.e., pH 7), deionized water, cisplatin hydrolyze tomonoaqua/monohydroxy platinum complexes, which is less likely to furtherhydrolyze to diaqua complexes. However, cisplatin can readily formmonoaqua and diaqua complexes by precipitation of chloro ligand withinorganic salts (e.g., silver nitrate, and the like). Also, the chloroligands can be replaced by existing nucleophile (e.g., nitrogen andsulfur electron donors, etc.) without undergoing aquation intermediates.

Cisplatin is relatively stable in human plasma, where a highconcentration of chloride prevents aquation of cisplatin. However, oncecisplatin enters a tumor cell, where a much lower concentration ofchloride exists, one or both of the chloro ligands of cisplatin isdisplaced by water to form an aqua-active intermediate form (as shownabove), which in turn can react rapidly with DNA purines (i.e., Adenineand Guanine) to form stable platinum—purine—DNA adducts.

Cisplatin enters the cell through both passive diffusion and activetransport. The pharmacological behavior of cisplatin is in partdetermined by hydrolysis reactions that occur once cisplatin is insidethe cell where the chloride concentration is essentially zero. In thisintracellular milieu, one chlorine ligand is replaced by a watermolecule to yield an aquated version of cisplatin. The aquated platinumcan then react with a variety of intracellular nucleophiles. Cisplatinbinds to RNA more extensively than to DNA and to DNA more extensivelythan to protein; however, all of these reactions are thought to occurintracellularly. Thus, upon administration, a chloride ligand undergoesslow displacement with water (an aqua ligand) molecules, in a processtermed aquation. The aqua ligand in the resulting [PtCl(H₂O)(NH₃)₂]⁺ iseasily displaced, allowing cisplatin to coordinate a basic site in DNA.Subsequently, the platinum cross-links two bases via displacement of theother chloride ligand. Cisplatin crosslinks DNA in several differentways, interfering with cell division by mitosis. The damaged DNA elicitsvarious DNA repair mechanisms, which in turn activate apoptosis whenrepair proves impossible. Most notable among the DNA changes are the1,2-intrastrand cross-links with purine bases. These include1,2-intrastrand d(GpG) adducts which form nearly 90% of the adducts andthe less common 1,2-intrastrand d(ApG) adducts. 1,3-intrastrand d(GpXpG)adducts may also occur, but are readily excised by the nucleotideexcision repair (NER) mechanism. Other adducts include inter-strandcrosslinks and nonfunctional adducts that have been postulated tocontribute to cisplatin's activity. In some cases, replicative bypass ofthe platinum 1,2-d(GpG) crosslink can occur allowing the cell tofaithfully replicate its DNA in the presence of the platinum cross link,but often if this 1,2-intrastrand d(GpG) crosslink is not repaired, itinterferes with DNA replication ultimately resulting in apoptosis.

The formation of cisplatin-DNA adducts that interfere with DNAreplication is illustrated in Scheme II:

Interaction with cellular proteins, particularly High Mobility Group(HMG) chromosomal domain proteins (which are involved withtranscription, replication, recombination, and DNA repair), has alsobeen advanced as a mechanism of interfering with mitosis, although thisis probably not its primary method of action. It should also be notedthat although cisplatin is frequently designated as an alkylating agent,it has no alkyl group and cannot carry out alkylating reactions.Accordingly, it is more accurately classified as an alkylating-likeagent.

Bu way of non-limiting example, the platinum compounds of the presentinvention include all compounds, compositions, and formulations whichcontaining a platinum ligand in the structure of the molecule. Thevalence of the platinum ligand contained therein may be platinum II orplatinum IV. The platinum medicaments of the present invention include,in a non-limiting manner, cisplatin, oxaliplatin, carboplatin,satraplatin, and analogs and derivatives thereof.

VII. Pharmacology of Formula (I) Compounds

The Formula (I) compounds, most notably for purposes of the presentinvention, dimesna (disodium-2,2′-dithiobis ethane sulfonate; BNP7787;Tavocept™ and the metabolite of dimesna, sodium-2-mercaptoethanesulfonate (mesna), act to selectively reduce the toxicity of certainantineoplastic agents in vivo. Mesna is utilized to reduce the acroleinrelated uroepithelial cell toxicity of ifosfamide and cyclophosphamide,and is currently approved for such usage in the United States andabroad.

Dimesna is the physiological auto-oxidation dimer of mesna. Mesna (I)and dimesna (II) have the following molecular structures:

The pharmaceutical chemistry of the compounds indicates that theterminal sulfhydryl group of mesna (and to a lesser extent the disulfidelinkage in dimesna) acts as a substitution group for the terminalhydroxy- or aquo-moiety in the active metabolites of platinum complexes.Dimesna, unlike mesna, requires a metabolic activation, such as byglutathione reductase, to exert its biologically efficacious results.Dimesna also exhibits significantly lower toxicity than mesna.

The conversion from the hydroxy- or aquo-moiety to a thioether isfavored, particularly under acidic conditions, and results in theformation of a hydrophilic compound of much lower toxicity, one which israpidly eliminated from the body.

Since blood plasma is slightly alkaline (pH ˜7.3), the more stabledisulfide form is the favored species, and does not readily react withthe nucleophilic terminal chlorine in cisplatin or the cyclobutanedicarboxylato moiety of carboplatin. This allows the drug to perform itsintended cytotoxic action on the targeted cancer cells. Postulated andhypothetical mechanisms of action for the platinum complexes arediscussed throughout the recent literature.

The compositions of the present invention comprise a therapeuticallyeffective amount of a Formula (I) compound. As previously defined, thecompounds of Formula (I) include pharmaceutically-acceptable salts ofsuch compounds, as well as prodrugs, analogs, conjugates, hydrates,solvates and polymorphs, stereoisomers (including diastereoisomers andenantiomers) and tautomers of such compounds. Compounds of Formula (I),and their synthesis are described in, e.g., U.S. Pat. Nos. 5,808,160,5,922,902, 6,160,167, and 6,504,049, the disclosures of which are herebyincorporated by reference in their entirety. In addition, Formula (I)compounds also include the metabolite of disodium 2,2′-dithio-bis-ethanesulfonate, known as 2-mercapto ethane sulfonate sodium (mesna) or2-mercaptoethane sulfonate as a disulfide form which is conjugated witha variety of substituent groups, as described in Published U.S. PatentApplication 2005/0256055, the disclosure of which is incorporatedherein, by reference, in its entirety.

The putative mechanisms of the Formula (I) compositions of the presentinvention which function in the potentiation of the anti-cancer activityof chemotherapeutic agents may involve one or more of several novelpharmacological and physiological factors, including but not limited to,a prevention, compromise, and/or reduction in the normal increase,responsiveness, or in the concentration and/or tumor protectivemetabolism of glutathione/cysteine and other physiological cellularthiols; these antioxidants and enzymes are increased in concentrationand/or activity, respectively, in response to the induction ofintracellular oxidative metabolism which may be caused by exposure tocytotoxic chemotherapeutic agents in tumor cells. Additional informationregarding certain mechanisms which may be involved in Formula (I)compounds is disclosed in U.S. patent application Ser. No. 11/724,933,filed Mar. 16, 2007, the disclosure of which is hereby incorporated byreference in its entirety.

Additionally, disclosure is provided herein which provides evidence thatFormula (I) compounds of the present invention also play a role in: (i)increasing patient survival time in cancer patients receivingchemotherapy; (ii) maintaining or stimulating hematological function inpatients in need thereof, including those patients suffering fromcancer; (iii) maintaining or stimulating erythropoietin function orsynthesis in patients in need thereof, including those patientssuffering from cancer; (iv) mitigating or preventing anemia in patientsin need thereof, including those patients suffering from cancer; (v)maintaining or stimulating pluripotent, multipotent, and unipotentnormal stem cell function or synthesis in patients in need thereof,including those patients suffering from cancer; (vi) promoting thearrest or retardation of tumor progression in those cancer patientsreceiving chemotherapy; and (vii) increasing patient survival and/ordelaying tumor progression while maintaining or improving the quality oflife in a cancer patient receiving chemotherapy.

Preferred doses of the Formula (I) compounds of the present inventionrange from about 1 g/m² to about 50 g/m², preferably about 5 g/m² toabout 40 g/m² (for example, about 10 g/m² to about 30 g/m²), morepreferably about 14 g/m² to about 22 g/m², with a most preferred dose of18.4 g/m².

VIII. Pharmacology of Erythropoietin and the Process of Erythropoiesis

Erythropoiesis is the process by which red blood cells (erythrocytes)are produced. In the early fetus, erythropoiesis takes place in themesodermal cells of the yolk sac. By the third or fourth month of fetaldevelopment, erythropoiesis moves to the spleen and liver. In humanadults, erythropoiesis generally occurs within the bone marrow. The longbones of the arm (tibia) and leg (femur) cease to be important sites ofhematopoiesis by approximately age 25; with the vertebrae, sternum,pelvis, and cranial bones continuing to produce red blood cellsthroughout life. However, it should be noted that in humans with certaindiseases and in some animals, erythropoiesis also occurs outside thebone marrow, within the spleen or liver. This is termed extramedullaryerythropoiesis.

In the process of red blood cell maturation, a cell undergoes a seriesof differentiations. The following stages of development all occurwithin the bone marrow: (i) pluripotent hematopoietic stem cell; (ii)multipotent stem cell; (iii) unipotent stem cell; (iv) pronormoblast;(v) basophilic normoblast/early normoblast; (vi) polychrmatophilicnormoblast/intermediate normoblast; (vii) orthochromic normoblast/latenormoblast; and (viii) reticulocyte. Following these stages, the cell isreleased from the bone marrow, and ultimately becomes an “erythrocyte”or mature red blood cell circulating in the peripheral blood. Thesestages correspond to specific histological appearances of the cell whenstained with Wright's stain and examined via light microscopy, but theyalso correspond to numerous other intrinsic biochemical andphysiological changes. For example, in the process of maturation, abasophilic pronormoblast is converted from a cell with a large nucleusand a volume of 900 μm³ to an enucleated disc with a volume of 95 μm³.By the reticulocyte stage, the cell has extruded its nucleus, but isstill capable of producing hemoglobin.

A feedback loop involving the cytokine glycoprotein hormoneerythropoietin (discussed below) helps regulate the process oferythropoiesis so that, in non-disease states, the production of redblood cells is equal to the destruction of red blood cells and the redblood cell number is sufficient to sustain adequate tissue oxygen levelsbut not so high as to cause blood thickening or “sludging”, thrombosis,and/or stroke. Erythropoietin is produced in the kidney and liver inresponse to low oxygen levels. In addition, erythropoietin is bound bycirculating red blood cells; low circulating numbers lead to arelatively high level of unbound erythropoietin, which stimulatesproduction in the bone marrow.

Recent studies have also shown that the peptide hormone hepcidin mayalso play a role in the regulation of hemoglobin production, and thuseffect erythropoiesis. Hepcidin, produced by the liver, controls ironabsorption in the gastrointestinal tract and iron release fromreticuloendothelial tissue. Iron must be released from macrophages inthe bone marrow to be incorporated into the heme group of hemoglobin inerythrocytes.

There are colony forming units (e.g., including the granulocyte monocytecolony forming units) that cells follow during their formation. Thesecells are referred to as the committed cells. For example, the loss offunction of the erythropoietin receptor or JAK2 in mice cells causesfailure in erythropoiesis, so production of red blood cells in embryosand growth is disrupted. Similarly, the lack of feedback inhibition,such as SOCS (Suppressors of Cytokine Signaling) proteins in the system,have been shown to cause gigantism in mice.

Erythropoietin (EPO) is a cytokine glycoprotein hormone that is acytokine for erythrocyte (red blood cell) precursors in the bone marrowwhich regulates the process of red blood cell production(erythropoiesis). Cytokines are a group of proteins and peptides thatfunction as signaling compounds produced by cells to communicate withone another. They act via cell-surface cytokine receptors. The cytokinefamily consists mainly of smaller water-soluble proteins andglycoproteins (i.e., proteins with an added sugar chain(s)) with a massof between 8 and 30 kDa. They act like hormones and neurotransmittersbut whereas hormones are released from specific organs into the bloodand neurotransmitters are produced by neurons, cytokines are released bymany types of cells. Due to their central role in the immune system,cytokines are involved in a variety of immunological, inflammatory, andinfectious diseases. When the immune system is fighting pathogens,cytokines signal immune cells such as T-cells and macrophages to travelto the site of infection. In addition, cytokines activate those cells,stimulating them to produce more cytokines. However, not all theirfunctions are limited to the immune system, as they are also involved inseveral developmental processes during embryogenesis. Cytokines areproduced by a wide variety of cell types (both hemopoietic andnon-hemopoietic), and can have effects on both nearby cells orthroughout the organism. Sometimes these effects are strongly dependenton the presence of other chemicals and cytokines. Cytokines may besynthesized and administered exogenously. However, such molecules can,at a latter stage be detected, since they differ slightly from theendogenous ones in, e.g., features of post-translational modification.

EPO is produced mainly by peritubular fibroblasts of the renal cortex.Regulation is believed to rely on a feed-back mechanism measuring bloodoxygenation. Constitutively synthesized transcription factors for EPO,known as hypoxia inducible factors (HIFs), are hydroxylized andproteosomally-digested in the presence of oxygen. See, e.g., Jelkmann,W. Erythropoietin after a century of research: younger than ever. Eur.J. Haematol. 78 (3):183-205 (2007). Hypoxia-inducible factors (HIFs) aretranscription factors that respond to changes in available oxygen in thecellular environment, in specific, to decreases in oxygen, or hypoxia.Most, if not all, oxygen-breathing species express the highly-conservedtranscriptional complex HIF-1, which is a heterodimer composed of an α-and a β-subunit, the latter being a constitutively-expressed arylhydrocarbon receptor nuclear translocator (ARNT).

HIF-1 belongs to the PER-ARNT-SIM (PAS) subfamily of the basichelix-loop-helix (bHLH) family of transcription factors. The α-subunitof HIF-1 is a target for propyl hydroxylation by HIF prolyl-hydroxylase,which makes HIF-1α a target for degradation by the E3 ubiquitin ligasecomplex, leading to quick degradation by the proteosome. This occursonly in normoxic conditions. In hypoxic conditions, HIFprolyl-hydroxylase is inhibited, since it utilizes oxygen as aco-substrate.

Hypoxia also results in a buildup of succinate, due to inhibition of theelectron transport chain in the mitochondria. The buildup of succinatefurther inhibits HIF prolyl-hydroxylase action, since it is anend-product of HIF hydroxylation. In a similar manner, inhibition ofelectron transfer in the succinate dehydrogenase complex due tomutations in the SDHB or SDHD genes can cause a build-up of succinatethat inhibits HIF prolyl-hydroxylase, stabilizing HIF-1α. This is termedpseudohypoxia.

HIF-1, when stabilized by hypoxic conditions, upregulates several genesto promote survival in low-oxygen conditions. These include glycolysisenzymes, which allow ATP synthesis in an oxygen-independent manner, andvascular endothelial growth factor (VEGF), which promotes angiogenesis.HIF-1 acts by binding to HIF-responsive elements (HREs) in promotersthat contain the sequence NCGTG. In general, HIFs are vital todevelopment. In mammals, deletion of the HIF-1 genes results inperinatal death. HIF-1 has been shown to be vital to chondrocytesurvival, allowing the cells to adapt to low-oxygen conditions withinthe growth plates of bones.

Erythropoietin is available as a therapeutic agent produced byrecombinant DNA technology in mammalian cell culture. It is used intreating anemia resulting from chronic kidney disease, from thetreatment of cancer (e.g., from chemotherapy and radiation) and fromother critical illnesses (e.g., heart failure).

In should be noted that there have been a number of recent warningsreleased by both pharmaceutical manufacturers and the United States Foodand Drug Administration (FDA) concerning the safety of EPO use in anemiccancer patients. Initially, a manufacturer of erythropoiesis-stimulatingagents (ESAs), disseminated a “Dear Doctor” letter in 2007, thathighlighted results from a recent clinical trial which examinedcancer-associated anemia, and warned doctors to consider use in thatoff-label indication with caution. An ESA manufacturer also advised theFDA regarding the results of three (3) clinical trials: the DAHANCA 10;PREPARE, and GOG-191 clinical trials. For example, DAHANCA refers to aseries of studies, entitled “Danish Head and Neck Cancer Studies” themost recent of which is “DAHANCA 10”. See. e.g., Eriksen, J. andOvergaard, J., Lack of prognostic and predictive value of CA IX inradiotherapy of squamous cell carcinoma of the head and neck with knownmodifiable hypoxia: An evaluation of the DAHANCA 5 study. Radiotherap.Oncol. 83(3):383-388 (2007). In this study, the DAHANCA 10 datamonitoring committee found that three year loco-regional control ofvarious types of head and neck cancers in subjects treated with an ESAwas significantly worse than for those not receiving an ESA (p=0.01). Inresponse to these advisories, the FDA subsequently released a PublicHealth Advisory and a clinical alert for physicians, regarding the useof ESAs. The advisory recommended caution in using these agents incancer patients receiving chemotherapy or off chemotherapy, andindicated a lack of clinical evidence to support improvements in qualityof life or transfusion requirements in these settings. In addition, ESAmanufacturers have agreed to new Black Box Warnings about the safety ofthese drugs. It should be noted that, additional information regardingvarious ESAs may be obtained from the Food and Drug Administration (FDA)or the specific ESA manufacturers themselves.

A related cytokine, colony-stimulating factors (CSF), are secretedglycoproteins which bind to receptor proteins on the surfaces ofhematopoietic stem cells and thereby activate intracellular signalingpathways which can cause the cells to proliferate and differentiate intoa specific kind of blood cell (typically white blood cells).Hematopoietic stem cells (HSC) are stem cells (i.e., cells retain theability to renew themselves through mitotic cell division and candifferentiate into a diverse range of specialized cell types) that giverise to all the blood cell types including myeloid (e.g., monocytes,macrophages, neutrophils, basophils, eosinophils, erythrocytes,megakaryocytes/platelets, dendritic cells, and the like) and lymphoidlineages (e.g., T-cells, B-cells, NK-cells, and the like). Thedefinition of hematopoietic stem cells has undergone considerablerevision in the last two decades. The hematopoietic tissue containscells with long-term and short-term regeneration capacities andcommitted multipotent, oligopotent, and unipotent progenitors. Recently,long-term transplantation experiments point toward a clonal diversitymodel of hematopoietic stem cells. Here, the HSC compartment consists ofa fixed number of different types of HSC, each withepigenetically-preprogrammed behavior. This contradicts older models ofHSC behavior, which postulated a single type of HSC that can becontinuously molded into different subtypes of HSCs. For example, HSCsconstitute 1:10.000 of cells in myeloid tissue.

Colony-stimulating factors may be synthesized and administeredexogenously. However, such molecules can at a latter stage be detected,since they differ slightly from endogenous ones in e.g.,post-translational modification. The name “colony-stimulating factors”comes from the method by which they were discovered. Hemopoietic stemcells were cultured on a so-called semi solid matrix which preventscells from moving around, so that if a single cell starts proliferating,all of the cells derived from it will remain clustered around the spotin the matrix where the first cell was originally located, and these arereferred to as “colonies.” It was therefore possible to add varioussubstances to cultures of hemopoietic stem cells and then examine whichkinds of colonies (if any) were “stimulated” by them. The substancewhich was found to stimulate formation of colonies of macrophages, forinstance, was called macrophage colony-stimulating factor, and so on.The colony-stimulating factors are soluble, in contrast to other,membrane-bound substances of the hematopoietic microenvironment. This issometimes used as the definition of CSF. They transduce by paracrine,endocrine, or autocrine signaling.

Colony-stimulating factors include: macrophage colony-stimulatingfactor; granulocyte-macrophage colony-stimulating factor; andgranulocyte colony-stimulating factor. Macrophage colony-stimulatingfactor (M-CSF or CSF-1), is a secreted cytokine which influenceshematopoietic stem cells to differentiate into macrophages or otherrelated cell types. M-CSF binds to the macrophage colony-stimulatingfactor receptor. It may also be involved in development of the placenta.

Granulocyte-macrophage colony-stimulating factor (GM-CSF or CSF-2), is aprotein secreted by macrophages, T-cells, mast cells, endothelial cells,and fibroblasts. GM-CSF is a cytokine that functions as a white bloodcell growth factor. GM-CSF stimulates stem cells to produce granulocytes(e.g., neutrophils, eosinophils, and basophils) and monocytes. Monocytesexit the circulation and migrate into tissue, whereupon they mature intomacrophages. It is thus part of the immune/inflammatory cascade, bywhich activation of a small number of macrophages can rapidly lead to anincrease in their numbers, a process crucial for fighting infection. Theactive form of the protein is found extracellularly as a homodimer.

Granulocyte Colony-Stimulating Factor (G-CSF or CSF-3), is acolony-stimulating factor hormone. It is a glycoprotein, growth factor,or cytokine produced by a number of different tissues to stimulate thebone marrow to produce granulocytes and stem cells. G-CSF thenstimulates the bone marrow to pulse them out of the marrow into theblood. It also stimulates the survival, proliferation, differentiation,and function of neutrophil precursors and mature neutrophils. G-CSF isproduced by endothelium, macrophages, and a number of other immunecells. The natural human glycoprotein exists in two forms, a 174- and180-amino acids-long protein of molecular weight 19,600 grams per mole.The more-abundant and more-active 174-amino acid form has been used inthe development of pharmaceutical products by recombinant DNA (rDNA)technology. The G-CSF receptor is present on precursor cells in the bonemarrow, and, in response to stimulation by G-CSF, initiatesproliferation and differentiation into mature granulocytes.Promegapoietin is a recombinant drug which is given during chemotherapyto increase blood cell regeneration. It is a colony-stimulating factorthat stimulates megakaryocyte production. It functions by stimulatingligands for interleukin-3 and c-Mpl.

IX. Mechanisms of Action of Tavocept™

An important element of Tavocept's™ effectiveness as a compound in thetreatment of cancer is its selectivity for normal cells versus cancercells and its inability to interfere with the anti-cancer activity ofchemotherapeutic agents. In vitro studies demonstrated that Tavocept™does not interfere with paclitaxel induced apoptosis, as assessed byPARP cleavage, Bcl-2 phosphorylation, and DNA laddering in human breast,ovarian and lymphoma cancer cell lines. Additionally, Tavocept™ did notinterfere with paclitaxel and platinum induced cytotoxicity in humancancer cell lines and did not interfere with paclitaxel and platinumregimens in the animals models discussed herein.

The potential mechanisms underlying the absence of interference withanti-cancer activity by Tavocept™ are multifactorial and, as previouslydiscussed, may involve its selectivity for normal cells versus cancercells, inherent chemical properties that have minimal impact in normalcells on critical plasma and cellular thiol-disulfide balances, and itsinteractions with cellular oxidoreductases, which are key in thecellular oxidative/reduction (redox) maintenance systems.

In addition to the absence of interference with anti-cancer activity,results from in vivo studies have shown that Tavocept™ may elicit therestoration of apoptotic sensitivity in tumor cells through thioredoxin-and glutaredoxin-mediated mechanisms and this may be an importantelement of its effectiveness as a chemotherapeutic agent. It has beendetermined that Tavocept™ is a substrate for thioredoxin and exhibitssubstrate-like activity with glutaredoxin in the presence of reducedglutathione and glutathione reductase, and this substrate-like activitymay be due to non-enzymatic formation of glutathione-containingdisulfide heteroconjugates during the assay reaction; these glutathionedisulfide heteroconjugates may, in turn, act as substrates forglutaredoxin. Thus, Tavocept™ could potentially shift the intracellularbalance of oxidized (inactive) and reduced (active) thioredoxin orglutaredoxin, subsequently modulating their cellular activity.

Similarly, increased concentrations of Tavocept™ cause a marked increasein the percent of inhibition of GST catalysis in the conjugation ofreduced glutathione to 1-chloro-2,4-dinitrobenzene (CDNB) (this datawill be presented, infra). One function of GST and related species(GSTs) is to protect mammalian cells against the neoplastic effects ofelectrophilic metabolites of carcinogens and reactive oxygen species by,e.g., catalyzing the conjugation of glutathione to a variety ofelectrophilic compounds. Moreover, GSTs are highly expressed in tumortissue relative to normal tissue, are found in high levels in the plasmaof cancer patients, and increased expression of GSTs has been linked tothe development of cellular resistance to alkylating cytostatic drugs.

Tavocept™ restoration of the apoptotic sensitivity of tumor cells viathioredoxin, glutaredoxin or related cellular redox systems, would havea net anti-proliferative activity on tumor cells. Thioredoxin and GSTare key players both in apoptotic pathways in cells and in theintracellular redox environment and any molecule that inhibits or servesas substrate for these proteins could offset changes in theintracellular redox environments that are due to high/elevated/aberrantlevels of thioredoxin and/or GST. The effect of Tavocept™ on thioredoxinand/or GST could also potentially normalize redox sensitive signalingpathways that are involved in apoptosis. Thus, the net results would bean increased sensitivity of tumor cells to chemotherapeutic agentsand/or restoration of a more normal intracellular redox environment Asubstantial increase in the inactive forms of these oxidoreductasescould result in significant changes in redox homeostasis, cellproliferation, and gene transcription through reductive control overvarious transcription factors. Specifically, the involvement of thethioredoxin system in tumor progression, its influence on p53-mediatedgene transcription, and its demonstrated roles in neuroprotectionagainst chemical toxins would indicate that interaction of this systemwith Tavocept™ could have a variety of positive clinical sequelaeincluding: (i) inhibition of tumor growth in the presence of oxidativestressors; (ii) protection of normal cells during chemically-inducedhyperoxidation and hyperthermia of cancer cells; and/or (iii)amelioration of chemically-induced neurotoxicity.

X. Activity of Tavocept™ on Physiological Cellular Thiols andNon-Protein Sulfhydryls (NPSH)

As the number of agents and treatments for cancer, as well as the numberof subjects receiving one or more of these chemotherapeutic agentsconcomitantly, has increased, clinicians and researchers are seeking tofully elucidate the biological, chemical pharmacological, and cellularmechanisms which are responsible for the pathogenesis andpathophysiology of the various adverse disease manifestations, as wellas how these chemotherapeutic drugs exert their anti-cancer andcytotoxic or cytostatic activity on a biochemical and pharmacologicalbasis. As described herein, with the exception of the novel conceptionand practice of the present invention, there is no pharmaceuticalcomposition(s) presently available which is: (i) is capable of affectingthe intracellular concentration of thioredoxin and glutaredoxin and/ormitigating or preventing thioredoxin- or glutaredoxin-mediatedresistance to chemotherapeutic agents results in an increase in cancerpatient survival time, in comparison to those cancer patients who didnot receive the pharmaceutical composition; and (ii) preventing ordelaying the initial onset of, attenuating the overall severity of,and/or expediting the resolution of the acute or chronic deleteriouschemotherapeutic agent-induced effects.

The mechanisms by which the Formula (I) compounds of the presentinvention (which include 2,2′-dithio-bis-ethane sulfonate andpharmaceutically-acceptable salts and analogs thereof) function involvesseveral novel pharmacological and physiological factors, including butnot limited to:

-   -   (i) a prevention, compromise and/or reduction in the normal        increase, responsiveness, or in the concentration and metabolism        of physiological cellular thiols; these antioxidants and enzymes        are increased in concentration and/or activity, respectively, in        response to the induction of changes in intracellular oxidative        metabolism which may be caused by exposure to chemotherapeutic        agents in tumor cells. The Formula (I) compounds of the present        invention exert an oxidative activity by the intrinsic        composition of the molecule itself (i.e., an oxidized        disulfide), as well as by oxidizing free thiols to form oxidized        disulfides (i.e., by non-enzymatic SN2-mediated reactions,        wherein attack of a thiol/thiolate upon a disulfide leads to the        departure of the more acidic thiol group. As the thiolate group        is far more nucleophilic than the corresponding thiol, the        attack is believed to be via the thiolate), and by the        pharmacological depletion and metabolism of reductive        physiological free thiols (e.g., glutathione, cysteine, and        homocysteine). These pharmacological activities will thus have        an additive effect on cytotoxic chemotherapy administration to        patients with cancer, and additional anti-cancer activity will        result from the administration of an oxidative        metabolism-affecting Formula (I) compound of the present        invention, increasing drug efficacy, and reducing the        tumor-mediated resistance of the various co-administered        chemotherapeutic agents, e.g., platinum, taxane, and alkylating        agent-based drug efficacy and tumor-mediated drug resistance;    -   (ii) thioredoxin inactivation by an oxidative        metabolism-affecting Formula (I) compound of the present        invention, thereby increasing apoptotic sensitivity and        decreasing mitogenic/cellular replication signaling in cancer        cells;    -   (iii) a key metabolite of the Formula (I) compound, Tavocept™        (disodium 2,2′-dithio-bis-ethane sulfonate), which is known as        2-mercapto ethane sulfonate sodium (also known in the literature        as mesna) possesses intrinsic cytotoxic or cytostatic activity        (i.e., causes apoptosis) in some tumors which can kill cancer        cells directly; and    -   (iv) it is believed that the Formula (I) compounds of the        present invention may act by causing changes in intracellular        oxidative metabolism of cancer tumor cells, and may enhance        their oxidative biological and physiological state and thereby        increase the amount of oxidative damage (e.g., mediated by ROS,        RNS or other mechanisms) in tumor cells exposed to chemotherapy,        thereby enhancing cytotoxicity/apoptosis of chemotherapy agents.        Thus, by altering intracellular oxidative metabolism by        enhancing levels of physiologically-deleterious oxidative        compounds and/or reducing or compromising the total        anti-oxidative capacity or responsiveness of cancer tumor cells,        a marked increase in anti-cancer activity can be achieved. It is        believed by the Applicant of the present invention that this is        a key mechanism of action (that may act in concert with various        other mechanisms of anti-cancer augmentation) of the Formula (I)        compounds of the present invention, with very important        implications for treatment.

Compositions and formulations comprising the Formula (I) compounds ofthe present invention may be given using any combination of thefollowing three general treatment methods: (i) in a direct inhibitory orinactivating manner (i.e., direct chemical interactions that inactivatethioredoxin and/or glutaredoxin) and/or depletive manner (i.e.,decreasing thioredoxin and/or glutaredoxin concentrations or productionrates), thereby increasing the susceptibility of the cancer cells to anysubsequent administration of any chemotherapeutic agent or agents thatmay act directly or indirectly through the thioredoxin- and/orglutaredoxin-mediated pathways in order to sensitize the patient'scancer and thus increase the survival of the patient; and/or (ii) in asynergistic manner, where the anti-thioredoxin and/or glutaredoxintherapy is concurrently administered with chemotherapy administrationwhen a cancer patient begins any chemotherapy cycle, in order toincrease and optimize the pharmacological activity directed againstthioredoxin- and/or glutaredoxin-mediated mechanisms present whilechemotherapy is being concurrently administered; and/or (iii) in apost-treatment manner (i.e., after the completion of chemotherapy doseadministration or a chemotherapy cycle) in order to maintain thepresence of a pharmacologically-induced depletion, inactivation, ormodulation of thioredoxin and/or glutaredoxin in the patient's cancercells for as long as optimally required. Additionally, theaforementioned compositions and formulations may be given in anidentical manner to increase patient survival time in a patientreceiving treatment with a cytotoxic or cytostatic anti-cancer agent byany additionally clinically-beneficial mechanism(s).

XI. Summary of Tavocept™-Related Studies Focusing on Potential Effectson the Thioredoxin and Glutaredoxin Systems

-   -   (i) Various Formula (I) compounds, including Tavocept™ (BNP7787,        dimesna) and Tavocept™-derived mesna disulfide heteroconjugates        function as alternative substrate inhibitors of the thioredoxin        and/or glutaredoxin systems (see, Tables 3 and 4; infra).    -   (ii) Various Formula (I) compounds, including Tavocept™ and        Tavocept™-derived mesna disulfide heteroconjugates have been        shown to promote formation of oxidized thioredoxin or oxidized        glutaredoxin, and since anti-apoptotic and cell growth signals        usually require reduced thioredoxin and reduced glutaredoxin,        this    -   (ii) Tavocept™-mediated shift towards oxidized thioredoxin        and/or glutaredoxin may result in increased apoptotic        sensitivity and inhibition of cell growth pathways.    -   (iii) Tavocept™ is a substrate (K_(m)=72 μM) for the coupled        thioredoxin/thioredoxin reductase system (but not thioredoxin        reductase alone).    -   (iv) Tavocept™ inhibits (K_(m)=3.6 mM) thioredoxin/thioredoxin        reductase catalyzed reduction of the insulin A-B chain        disulfide.    -   (v) Tavocept™ may depleted intracellular glutathione resulting        in formation of a Tavocept™-derived mesna disulfide        heteroconjugates (e.g., BNP7772). Tavocept™ is believed to        interfere with glutathione-mediated reduction of oxidized        glutaredoxin by serving as an alternative substrate inhibitor of        reduced glutaredoxin and/or by depleting intracellular        glutathione available to reduce oxidized glutaredoxin to the        active reduced form.

A better understanding of the present invention will be gained byreference to the following section disclosing Specific Examples andExperimental/Clinical Results. The following examples are illustrativeand are not intended to limit the invention or the claims in any manner.

Specific Examples and Experimental/Clinical Results I. Effects ofTavocept™ on Glutathione-S-Transferase (GST)

One potential hypothesis set forth to explain the ability of Tavocept™(disodium 2,2′-dithio-bis-ethane sulfonate; BNP7787) to augment theanti-cancer activity of chemotherapeutic agents states that Tavocept™may act as a glutathione surrogate or modulator in the reactions ofglutathione-S-transferase (GST). Glutathione and its related enzymesplay a major role in the detoxification of toxic chemicals includingcytotoxic chemotherapeutics. Glutathione-S-transferases (GSTs)constitute a family of phase II detoxifying isozymes that catalyze theconjugation of glutathione to a variety of electrophilic compounds,often the first step in the formation of mercapturic acid derivativessuch as N-acetylcysteine. Reaction Scheme I, below, illustratesGlutathione S-transferase catalyzing the transfer of glutathione to anelectrophilic species RX (wherein, R is S, N or C).

The resulting glutathione conjugates are either excreted from the cellor they undergo further enzymatic processing by γ-glutamyltranspeptidase and cysteine-S-conjugate-β-lyase. See, e.g., Hausheer, F.H., et al., Modulation of platinum-induced toxicities and therapeuticindex: mechanistic insights and first- and second-generation protectingagents. Semin. Oncol. 25:584-599 (1998). Glutathione-S-transferases(GSTs) are highly expressed in tumor tissue relative to normal tissuesand are also found in high levels in the plasma of cancer patients;thereby making these enzymes useful as potential cancer markers. Thereare multiple cytosolic- and membrane-bound GST isozymes that differ intheir tissue-specific expression and distribution. GSTs protectmammalian cells against the toxic and neoplastic effects ofelectrophilic metabolites of carcinogens and reactive oxygen species.For example, increased expression of GSTs has been linked to thedevelopment of cellular resistance to alkylating cytostatic drugs. Adeficiency of GST isozymes may increase the predisposition to variousforms of cancer. Therefore, GST status may be a useful diagnostic factorin determining the clinical outcome of chemotherapy.

The following experiments were designed to determine if Tavocept™ has aninhibitory or stimulatory effect on GST. Specifically, these studiesaddress whether Tavocept™ can act as a substrate for GST or if either ofthese compounds inhibit GST. An in vitro assay for GST has beendeveloped and reported. See, Meyer, D. J. and Ketterer, B., Purificationof soluble human glutathione S-transferases. Methods Enzymol. 252:53-65(1995). This assay monitors the conjugation of reduced glutathione to1-chloro-2,4-dinitrobenzene (CDNB), as illustrated in Reaction SchemeII, below.

Reduced thiol forms a conjugate with CDNB (extinction coefficient=9600M⁻¹ cm⁻¹), which is detected at 340 nm. Stock solutions of GSH, CDNB,Tavocept™ were prepared by dissolving the reagent in sterile water atthe concentrations listed below prior to use. A typical 1 mL assay wasset up by mixing 500 μl NaHPO₄ buffer (200 mM, pH 6.5), 20 μL GSH (50mM), 20 μL CDNB (50 mM), and 458 μL sterile water. Reactions wereincubated at 20° C. in the cuvette holder of the spectrophotometer forapproximately 5 min. prior to initiating the assay with the addition ofenzyme (ml−1 isotype of GST; activity >100 U/mg). The enzyme stockpurchased from the vendor was diluted 1:100 in 200 mM NaHPO₄ buffer (pH6.5), and 2 μL of the diluted enzyme was added to initiate the reaction.The final amount of enzyme added to the assay was typically 0.002 U.Assays were run at 20° C. in 1 mL quartz cuvettes (Hellma Scientific).Slopes were measured in the linear range of the assay (i.e., typicallybetween 5 to 10 min.). In assays where the effect of Tavocept™ on GSTactivity was measured, 20 μL of either a 500 mM, 166.7 mM, or 55.6 mMstock solution of Tavocept™ was added to standard reactions using 1 mMGSH as the enzyme substrate. Final reaction volumes were fixed at 1 mLby adjusting the amount of water added.

a. All UV-visible assays were performed using a Varian Cary 100spectrophotometer equipped with a thermostatic jacketed multi-cellholder. The default parameters of the Cary Win UV Enzyme Kineticsapplication (version 2.00) were used; with the exceptions of using boththe visible and deuterium lamps, and setting the wavelength to 340 nm,the temperature to 20° C., and the maximal duration of the assay at 30minutes.

Raw data was obtained on a Cary 100 spectrophotometer. This data showedseveral phases to a typical reaction. The first phase was a baselinecorresponding to the time prior to addition of enzyme (typically 2-5min. in duration). Assays in the first phase of the reaction containedonly substrate, buffer and (in some assays) Tavocept™. Thespectrophotometer was put in pause mode while enzyme (GST) was added andmixed into the assay reactions. No absorbance values were collectedduring the process of enzyme addition. The region of experimentalinterest was during the linear phase of the enzyme reaction, whichimmediately followed the addition of enzyme. The linear phase is ofexperimental interest because it is when the classical model ofMichaelis-Menton kinetics holds true. During this phase the substrateconcentration is high (>Km for enzyme) and, therefore, the rate ofcatalysis is independent of the substrate concentration. It was duringthis time that reaction rates (i.e., slopes of change in absorbance withtime) were measured using the Cary 100 software. The duration of thelinear phase was between 5-10 minutes, depending upon the specificreaction conditions. Reactions were considered complete when substrateconcentration was no longer saturating and became a rate limiting factorof the assay. When the substrate was limiting, the reaction ratedeviated from linearity. This end phase of the reaction was typicallyobserved after 10 to 15 minutes. Absorbance and time values during theend phase of the reaction were not used in slope calculations becausethe reaction was effectively over at this point as the reaction nolonger followed the classical Michaelis-Menton model for enzymekinetics. Completion of the reaction on the Cary software could bedetected visually by overlaying a straight line beginning at theaddition of enzyme and extending past the end phase of the assay curve.Upon completion of a set of reactions data was stored as an electronic“batch” file. Sigma Plot was used specifically to show the mean ofassays run in triplicate with linear regression lines and error barsillustrating standard deviation. Descriptive statistics (mean andstandard deviation) were used to describe and summarize the results ofthe experiments. The results of these experiments are illustrated inTable 2, below.

The GST reaction was performed in the presence of Tavocept™. FinalTavocept™ concentrations are shown to the right of each regressioncurve. Data points shown represents the average curve of triplicateexperiments for each assay condition, and error bars are standarddeviation. Assays were measured after the addition of GST in the linearrange (i.e., 8.9 min. to 13.1 min.).

The individual slopes for each of the three assay runs for a givenTavocept™ concentration, the standard deviation, the mean, the relativeenzyme activity, and percent inhibition are listed in Table 2, below.

TABLE 3 Rates of GST Assays Run in the Presence of Tavocept™ Tavocept™Slope Standard · Slope Relative Percent Concentration Abs/min DeviationMean Activity Inhibition 0 mM 0.0465 0.0029 0.0449  100% 0  0 mM 0.04240.0023 0 mM 0.0458 0.0023 1.1 mM 0.0427 0.0023 0.0424 94.4%  5.6 1.1 mM0.0437 0.0020 1.1 mM 0.0407 0.0020 3.3 mM 0.0295 0.0014 0.0274   61%39   3.3 mM 0.0242 0.0009 3.3 mM 0.0284 0.0011 10 mM 0.0155 0.00120.0151 33.6% 66.4 10 mM 0.0158 0.0012 10 mM 0.0139 0.0009

Table 3 shows the slopes for each assay trial, which were calculatedfrom the change in absorbance at 340 nm per minute in the linear portionof the assay. In these examples, the slope was measured from 8.9 to 13.1min. The relative activity was normalized using the slope mean to thereactions having no Tavocept™ added; and percent inhibition wascalculated as the difference of relative activity from 100%.

Accordingly, the data obtained from both Table 2 and Table 3 illustratethat increased concentrations of Tavocept™ cause a marked increase inthe percent of inhibition of GST catalysis in the conjugation of reducedglutathione to 1-chloro-2,4-dinitrobenzene (CDNB), as initiallyillustrated in Reaction Scheme II, above. For example, an increase ofTavocept™ from 1.1 mM to 3.3 mM was shown to cause an increase in thepercent inhibition from 5.6% to 39.0%. Thus, this relatively smallincrease in Tavocept™ concentration caused an approximate 6-timesincrease in GST inhibition.

One function of GST and related species (GSTs) is to protect mammaliancells against the neoplastic effects of electrophilic metabolites ofcarcinogens and reactive oxygen species by, e.g., catalyzing theconjugation of glutathione to a variety of electrophilic compounds.Moreover, GSTs are highly expressed in tumor tissue relative to normaltissues, are found in high levels in the plasma of cancer patients, andincreased expression of GSTs has been linked to the development ofcellular resistance to alkylating cytostatic drugs. Thus, it is probablethat one possible mechanism of action of Tavocept™ may be to cause achange or changes in the intracellular oxidative metabolism (i.e., theoxidative/reductive potential) within tumor cells so as to increase theintracellular levels of physiologically-deleterious oxidative compounds.This change may, in turn, cause the tumor cell to exhibit greatersensitivity to a chemotherapeutic agent without directly affecting themechanism of action of the chemotherapeutic agent itself.

II. Effects of Formula (I) Compounds on the Coupled GRX/GSH/GR System

FIG. 1 illustrates the involvement of (reduced) glutaredoxin inpromoting cell growth and/or stimulating cell proliferation via severalmetabolic pathways. The glutaredoxin system consists of glutaredoxin,glutathione and glutathione reductase. It should be noted, however, thatglutaredoxin is also involved in many other intracellular pathways. FIG.2 illustrates the coupled glutaredoxin (GRX)/glutathione(GSH)/glutathione reductase (GR) system.

Table 4, below, illustrates that various Formula (I) compounds (i.e.,dithiol-containing compounds) may act as alternative substrateinhibitors for the coupled GRX/GSH/GR system as measured by NADPHoxidation. The Formula (I) compound was utilized at a concentration of0.5 mM.

TABLE 4 NADPH Oxidation (nmoles/min/mL)^(1, 2) Disulfide (0.5 mM)³Thioredoxin Reductase only Thioredoxin Reductase + Thioredoxin BNP77870.3 ± 0.01 13.1 ± 0.2 BNP7772 (GSSM) 0.3 ± 0.02 14.1 ± 0.1 BNP7766(CSSM) 0.2 ± 0.03 14.4 ± 0.2 BNP7768 (HSSM) 0.0 ± 0.03  8.6 ± 0.06BNP7774 (ECSSM) 0.3 ± 0.02  9.6 ± 0.2 BNP7776 (GlyCSSM) 0.2 ± 0.4  15.8± 0.3 ¹Oxidation rates calculated from a minimum of triplicate assays.²A two-way ANOVA analysis was performed on the whole dataset. Thedifference rates for type A reactions and type B reactions wasstatistically significant (p = .0001), and was affected by the disulfideused (p = .0001). ³Rates calculated from positive absorbance changes orabsorbance changes of less than .0001 are shown as 0.0.

III. Effects of Formula (I) Compounds on the Coupled TX/TXR System

The TX system plays an important role in the redox regulation of anumber of cellular processes, notably modulation of apoptosis andcellular proliferation. The system includes the selenoprotein,thioredoxin reductase (TXR), and its main substrate, thioredoxin (TX),as well as thioredoxin peroxidase (TPX). See, e.g., Zhong, L., et al.,Rat and calf thioredoxin reductase are homologous to glutathionereductase with a carboxyl-terminal elongation containing a conservedcatalytically active penultimate seloncysteine residue. J. Biol. Chem.273: 8581-8591, 1998 Holmgren, A. Thioredoxin and glutaredoxin systems.J. Biol. Chem. 264:13963-13966 (1989). TXR is a pyridinenucleotide-disulfide oxidoreductase, and catalyzes the NADPH-dependentreduction of the active site disulfide in oxidized thioredoxin (see,Reaction Scheme III; TRX-S₂) to give a dithiol in reduced thioredoxin(TX-(SH)₂). See, e.g., Zhong, L., et al. Rat and calf thioredoxinreductase are homologous to glutathione reductase with acarboxyl-terminal elongation containing a conserved catalytically activepenultimate seloncysteine residue. J. Biol. Chem. 273:8581-8591 (1998).Reaction Scheme III, below, outlines the various reaction mechanismsinvolved in the TX redox regulation system.

Reaction Scheme III

NADPH+H⁺+TX-S₂→TX-(SH)₂+NADP⁺

XSSY+TX-(SH)₂→TX-S₂+XSH+YSH

TPX-S₂+TX-(SH)₂→TX-S₂+TPX-(SH)₂

H₂O₂+TPX-(SH)₂→TPX-S₂+H₂O

TX is a small disulfide reductase with a broad range of substrates andimportant functions in the redox modulation of protein signaling and thereductive activation of a number of important transcription factors.See, e.g., Welsh, S. J., et al., The thioredoxin redox inhibitors1-methylpropyl 2-imidazolyl disulfide and pleurotin inhibithypoxia-induced factor 1 alpha and vascular endothelial growth factorformation. Mol. Cancer Therapy 2:235-243 (2003). Like glutaredoxin(GRX), TX is only active in its reduced form (TX-(SH)₂) which serves asa hydrogen donor for ribonucleotide reductase and other redox enzymes,and acts in defense against changes in intracellular oxidativemetabolism. While they share some substrate specificity, the TX systemis more catalytically diverse than the GRX system and does not interactsubstantially with glutathione (GSH). See, e.g., Luthman, M., andHolmgren, A. Rat liver thioredoxin and thioredoxin reductase:purification and characterization. Biochemistry 21:6628-6633 (1982).

FIG. 3 illustrates several representative thioredoxin-related pathwaysinvolved in cell proliferation and apoptosis. For thioredoxin (TX) topromote cell growth, inhibit apoptosis or stimulate cell proliferation,it must be in the reduced form. It should be noted, however, that TX isalso involved in many other intracellular pathways. FIG. 4 illustratesthe coupled thioredoxin (TX)/thioredoxin reductase (TXR) system.

The objective of the following experimental study was to determine ifTavocept™ has a detectable, direct interaction with the followingoxidoreductase enzymes: glutathione reductase (GR); glutaredoxin (GRX);glutathione peroxidase (GPX); thioredoxin reductase (TXR); andthioredoxin (TX). Based upon the nature and magnitude of theinteraction, it may be determined whether an interaction with redoxbalance enzymes could serve to explain clinical findings regardingTavocept™ metabolism or its mechanism of action.

The activity of TXR and TX was determined by following NADPH oxidationat 340 nm according to the previously reported method. See, Luthman, M.,and Holmgren, A. Rat liver thioredoxin and thioredoxin reductase:purification and characterization. Biochemistry 21:6628-6633 (1982). Atypical assay mixture contained TR buffer (50 mM potassium phosphate, pH7.0, 1 mM EDTA), 200W NADPH, 1.6 μg bovine TX, and one or more of thefollowing: 4.8 μM TXR, 86 μM insulin, and one of the disulfidesdescribed herein. All disulfides were added to reactions as 10×solutions in TR buffer. The total volume of each reaction was 0.1 mL.Reactions were initiated by the addition of TX and were incubated at 25°C. for 40 min. The activity was calculated using a 4 min. linear portionof each reaction. Enzyme assays were carried out using either aMolecular Devices SpectraMaxPlus UV plate reader or a Varian Cary 100UV-visible Spectrophotometer.

Data was then collected and plotted in Microsoft Excel. Errorcalculations, and graphical representations were performed in MicrosoftExcel and Kaleidograph (ver. 3.5). Nonlinear data was graphicallyrendered using Kaleidograph. ANOVA and other statistical analyses wereperformed using SAS (ver. 8.2). Unless otherwise noted, significancelevel was set at 0.05, and error bars represent actual experimentalstandard deviation.

The activity of TXR and TX with Tavocept™ is depicted in Table 5, below.Tavocept™ causes a concentration-dependent increase in NADPH oxidationby TXR in the presence of TX. In the absence of TX, the NADPH oxidationby TXR is indistinguishable from background. Based upon the magnitudeand concentration-dependence of the observed oxidation responses,Tavocept™ is most likely a substrate for TX, but not for TXR. It shouldbe noted that for the purposes of Table 5 only, thioredoxin is labeledTXR and thioredoxin reductase is labeled TRR.

Table 6, below, illustrates that various Formula (I) compounds (i.e.,disulfide-containing compounds) of the present invention can serve asalternate substrate inhibitors for the coupled thioredoxin(TX)/thioredoxin reductase (TXR)/NADPH system as measured by theoxidation of NADPH. In Table 6, the Formula (I) compounds were utilizedat a concentration of 0.5 mM.

TABLE 6 NADPH Oxidation (nmoles/min/mL)^(1, 2) Disulfide (0.5 mM) GRGR + GSH GR + GRX + GSH BNP7787 (MSSM)  0.0 ± 0.01  2.9 ± 1.6 15.3 ± 1.0BNP7772 (GSSM) 8.0 ± 0.6 11.3 ± 0.8 71.0 ± 7.9 BNP7766 (CSSM)  0.0 ±0.01  4.1 ± 1.3 28.3 ± 2.0 BNP7768 (HSSM) 0.16 ± 0.96 0.88 ± 0.2 10.7 ±0.7 BNP7774 (ECSSM) 0.04 ± 0.12  2.4 ± 0.7 37.0 ± 2.1 BNP7776 (GCSSM)0.0 ± 0.7  4.1 ± 1.0 22.0 ± 0.5 BNP7774S (ECSSCE)  0.1 ± 0.05  2.1 ± 0.222.4 ± 1.7 BNP7776S (GCSSCG) 0.0 ± 0.5  1.6 ± 0.6 15.3 ± 0.4 ¹Rates wereaverage of least two separate experiments in triplicate (n = 6).²Two-way ANOVA analysis of the whole dataset shows that A, B, and Crates are significantly different among the disulfides tested (p-value =.001). One-way ANOVA analyses for each disulfide show that (1) oxidationrates in the presence of GRX (reaction C conditions) were significantlyincreased, and (2) Rates in reaction B conditions were significantlyincreased for all disulfides except GSSM and HSSM. ³Absorbance changesof less than .0005 were assigned as 0.0. ⁴BNPXXXX refer to BioNumerikPharmaceuticals, Inc. proprietary compounds which all contain adisulfide moiety (SS).

IV. Summary of Tavocept™-Related Studies on the TX and GRX Systems

Various experimental data indicates that Tavocept™ (BNP7787, dimesna)and Tavocept™-derived mesna disulfide heteroconjugates formed as aconsequence of thiol-disulfide exchange reactions may interact with thethioredoxin (TX) and glutaredoxin (GRX) systems in the following ways:

-   -   1) Tavocept™ drives the oxidation of reduced thioredoxin to        oxidized thioredoxin;    -   2) BNP7787 derived metabolites (BNP7772, BNP7766, BNP7768,        BNP7774 and BNP7776) are substrates (i.e., alternative substrate        inhibitors) for the coupled thioredoxin/thioredoxin        reductase/NADPH (see, FIG. 3, FIG. 5, and Table 1);    -   3) Tavocept™ inhibits the TX/TXR catalyzed reduction of the        insulin A-B chain disulfide bond (and could inhibit reduction of        other protein disulfides by TX/TXR interfering with signaling        pathways);    -   4) Although Tavocept™ is not a substrate for glutathione        reductase (the enzyme that reduces the disulfide form of        glutaredoxin), the Tavocept™ metabolite BNP7772 (a        Tavocept™-derived mesna-disulfide heteroconjugate) functions as        an alternative substrate inhibitor and as such may compete with        the GR catalyzed reduction of glutathione disulfide. This could        inhibit glutaredoxin related signaling and cell proliferation        pathways (see, FIG. 1, FIG. 2; and Table 4);    -   5) The Tavocept™ metabolite, mesna, in combination with        cisplatin enhanced the reduction of α-lipoic acid (TX/TXR        substrate) or hydroxyethyldisulfide (GRX substrate) by whole        cells and intracellularly this mesna/cisplatin effect is        predicted to result in a shift in equilibrium towards oxidized        thioredoxin and glutaredoxin); and    -   6) Whole cell mediated disulfide reduction declined in response        to treatments with paclitaxel, cisplatin and Tavocept™, and        intracellularly this could be coupled with an altered redox        balance favoring oxidized thioredoxin and oxidized glutaredoxin.        This altered redox state would be expected to result in        increased apoptotic sensitivity and decreased cell        proliferation.

V. Summary of Tavocept™-Related Cytotoxicity Studies in Human CancerCell Lines

-   -   1) In non-small cell lung carcinoma (NSCLC) cell lines, mesna        (100 μM) in combination with paclitaxel enhanced the cytotoxic        effect of paclitaxel in comparison to paclitaxel alone controls;    -   2) In NSCLC and ovarian cancer cell lines, mesna (100 μM) in        combination with oxaliplatin markedly enhanced the cytotoxic        effect of oxaliplatin in comparison to oxaliplatin alone        controls. In this same study, a lesser increase in oxaliplatin        cytotoxicity was observed in brain cancer cells that were        treated with oxaliplatin and mesna; this effect in brain cancer        cells was observable but not statistically significant; and    -   3) In NSCLC and breast cancer cell lines, Tavocept™ in        combination with cisplatin resulted in an increase in cell death        in comparison to cisplatin only controls.

VI. Japan Phase III Clinical Trial A. Summary of the Objectives andMethods of the Japan Phase III Clinical Trial

Data was recently unblinded from a multicenter, double-blind,randomized, placebo-controlled Phase III clinical trial of the Formula(I) compound Tavocept™ (also known as BNP7787, disodium2,2′-dithio-bis-ethane sulfonate, and dimesna) conducted in Japan andinvolving patients with advanced non-small cell lung carcinoma (NSCLC),including the adenocarcinoma sub-type, who received the chemotherapeuticdrugs paclitaxel and cisplatin (for purposes of this document referredto as the “Japan Phase III Clinical Trial”).

The primary objective of the Japan Phase III Clinical Trial was to showthat the Formula (I) compound, Tavocept™, prevents and/or reducesperipheral neuropathy induced by paclitaxel+cisplatin combinationtherapy in patients with non-small cell lung carcinoma (NSCLC),including the adenocarcinoma sub-type.

Patients admitted into the trial included those patients withoutprevious treatment (excluding surgical treatment, administration ofPicibanil into the serous membrane, irradiation of 30% or lesshematopoietic bone, or oral chemotherapeutic agents within 3 months ofentry in the trial).

The Japan Phase III Clinical Trial was conducted as a double-blind studybecause peripheral neuropathy is diagnosed based on subjective symptomsevaluated through clinical interviews, lab tests, and the like.Accordingly, evaluations by both physicians and patients are highlyimportant. The present trial was designed to show that Tavocept™prevents and/or reduces peripheral neuropathy induced by paclitaxel andcisplatin in NSCLC patients, including the adenocarcinoma sub-type. Aplacebo was used as control since there is no established therapy ordrug for preventing peripheral neuropathy. Because the severity ofperipheral neuropathy is evaluated based on patient's reports (i.e.,subjective symptoms), the Peripheral Neuropathy Questionnaire (PNQ©) wasused in primary evaluation. CIPN-20 and NCI-CTC were used in secondaryevaluation. The incidence and severity of adverse reactions, time totheir onset, etc. and the like, were compared between patients treatedwith Tavocept™ and those given a placebo using the aforementionedmethods.

In order to conduct the present trial, Tavocept™ (approximately 14-22g/m², most preferably approximately 18.4 g/m²) or placebo (0.9% NaCl)was administered to NSCLC, including the adenocarcinoma sub-type,patients receiving chemotherapy with paclitaxel (approximately 160-190mg/m², most preferably approximately 175 mg/m²) and cisplatin(approximately 60-100, most preferably approximately 80 mg/m²), every 3weeks (and repeated for a minimum of 2 cycles).

B. Summary of the Results of the Japan Phase III Clinical Trial

The Japan Phase III Clinical Trial data demonstrated medically-importantreductions in chemotherapy-induced peripheral neuropathy for patientsreceiving Tavocept™ and chemotherapy compared to patients receivingchemotherapy and a placebo. In addition, there were concurrentobservations in the clinical trial population of medically-importantreductions in chemotherapy-induced vomiting/emesis and kidney damage.

The aforementioned clinical trial also provided a number of unexpectedphysiological results which have, heretofore, been unreported in anyprevious scientific or clinical studies. Importantly, the Japan PhaseIII Clinical Trial demonstrated increased survival times for patientswith advanced non-small cell lung cancer (NSCLC) receiving Tavocept™ andchemotherapy. A medically-important increase in survival time was alsoobserved in patients with the NSCLC adenocarcinoma sub-type receivingTavocept™ and chemotherapy. In addition, these unexpected and novelresults included, but were not limited to, (i) the differentiation ofchemotherapy-induced peripheral neuropathy into an entirely new class ofperipheral neuropathy, called “intermittent” or “sporadic” peripheralneuropathy; (ii) potentiation of the cytotoxic or apoptotic activitiesof chemotherapeutic agents in patients with non-small cell lungcarcinoma (NSCLC), including the adenocarcinoma sub-type, receivingTavocept™ and chemotherapy; (iii) increasing patient survival and/ordelaying tumor progression while maintaining or improving the quality oflife in patients with non-small cell lung carcinoma (NSCLC), includingthe adenocarcinoma sub-type, receiving Tavocept™ and chemotherapy; and(iv) the maintenance or stimulation of hematological function (e.g., anincrease in hemoglobin, hematocrit, and erythrocyte levels), in patientswith non-small cell lung carcinoma (NSCLC), including the adenocarcinomasub-type, receiving Tavocept™ and chemotherapy.

FIG. 5 illustrates, in tabular form, the Primary Endpoint (i.e., themitigation or prevention of patient peripheral neuropathy) of the JapanPhase III Clinical Trial supporting the present invention as determinedutilizing the Peripheral Neuropathy Questionnaire) (PNQ©). Resultsillustrated in FIG. 5 demonstrate that there was an approximate 50%reduction in severe (Grade D or E) peripheral neuropathy in the patientpopulation with non-small cell lung carcinoma (NSCLC), including theadenocarcinoma sub-type, who were treated with apaclitaxel/Tavocept™/cisplatin regimen in comparison to those patientswho received a paclitaxel/saline placebo/cisplatin regimen.

FIG. 6 illustrates, in tabular form, an evaluation of the statisticalpower observed in the Japan Phase III Clinical Trial with respect to thePrimary Endpoint (i.e., the mitigation or prevention of patientperipheral neuropathy), as measured by the Generalized EstimatingEquation (GEE) statistical method. The numerical value of 0.1565 in thetabular row designated “Drug” under the tabular column designated“P-Value” in FIG. 6, demonstrates that there is only a 15.65%probability that the reduction in peripheral neuropathy observed forTavocept™ in the Japan Phase III Clinical Trial is due to random chancealone.

FIG. 7 illustrates, in tabular form, a Secondary Endpoint (i.e., adecrease in patient hemoglobin, erythrocyte, and hematocrit levels) ofthe Japan Phase III Clinical Trial supporting the present invention, inpatients receiving Tavocept™ and chemotherapy. Results illustrated inFIG. 7 demonstrate that only 2, 1, and 1 non-small cell lung carcinoma(NSCLC), including the adenocarcinoma sub-type, patients in theTavocept™ arm of the study exhibited a Grade 3 (severe) decrease inhemoglobin, red blood cell, and hematocrit levels, respectively, incomparison to 8, 5, and 5 patients in identical categories in theplacebo arm of the Japan Phase III Clinical Trial.

FIG. 8 illustrates, in tabular form, a Secondary Endpoint (i.e., tumorresponse rate to chemotherapy administration) of the Japan Phase IIIClinical Trial supporting the present invention, in patient populationsreceiving either Tavocept™ or placebo, as measured by the physician orby the Independent Radiological Committee (IRC) criteria. As is shown inthe portion of the table designated “Doctor”, the Response Rate, asmeasured by physicians, in the Tavocept™ arm of the Japan Phase IIIClinical Trial was 41.9% compared to a 33.0% Response Rate in theplacebo arm. As shown in the portion of the table designated “IRC”, theresponse rate as measured by the IRC in the Tavocept™ arm of the JapanPhase III Clinical Trial was 33.3% as compared to a 28.6% response ratein the placebo arm.

FIG. 9 illustrates, in graphical form, a Secondary Endpoint (i.e.,patient survival) of the Japan Phase III Clinical Trial supporting thepresent invention, in patient populations receiving either Tavocept™ orplacebo. Results illustrated in FIG. 9 demonstrate an increase in mediansurvival time of up to 40 days in the portion of the patient populationwith non-small cell lung carcinoma (NSCLC), including the adenocarcinomasub-type, who were treated with a paclitaxel/Tavocept™/cisplatin regimenin comparison to median survival time for those patients who received apaclitaxel/saline placebo/cisplatin regimen.

FIG. 10 illustrates, in graphical form, a Secondary Endpoint (i.e.,patient survival) of the Japan Phase III Clinical Trial supporting thepresent invention, in female patient populations receiving eitherTavocept™ or placebo. Results in FIG. 10 demonstrate that the portion ofthe female patient population with non-small cell lung carcinoma(NSCLC), including the adenocarcinoma sub-type, who were treated with apaclitaxel/Tavocept™/cisplatin regimen had a longer survival period incomparison to the female patient population who received apaclitaxel/saline placebo/cisplatin regimen.

FIG. 11 illustrates, in graphical form, a Secondary Endpoint (i.e.,patient survival) of the Japan Phase III Clinical Trial supporting thepresent invention, in patient populations diagnosed with theadenocarcinoma sub-type of non-small cell lung carcinoma (NSCLC)receiving either Tavocept™ or placebo. Results illustrated in FIG. 11demonstrate an increase in median survival time of up to 138 days in theportion of the patient population with adenocarcinoma who were treatedwith a paclitaxel/Tavocept™/cisplatin regimen in comparison to themedian survival time for those patients who received a paclitaxel/salineplacebo/cisplatin regimen.

In addition, results from the Japan Phase III Clinical Trial alsodemonstrated reductions in: (i) fatigue (p=0.0163); (ii) nausea/vomiting(p=0.0240); (iii) anorexia (p=0.0029); (iv) diarrhea (p=0.0859); (v)constipation (p=0.1114); and (vi) insomnia (p=0.1108) in the portion ofthe patient population with non-small cell lung carcinoma (NSCLC) whowere treated with a paclitaxel/Tavocept™/cisplatin regimen in comparisonto those NSCLC patients who received a paclitaxel/salineplacebo/cisplatin regimen.

The results from the Japan Phase III Clinical Trial described in theinstant application represent medically important developments thatsupport surprising new findings for Formula (I) compounds, includingpotential uses for: (i) increasing patient survival time in cancerpatients receiving chemotherapy; (ii) causing cytotoxic or apoptoticpotentiation of the anti-cancer activity of chemotherapeutic agents incancer patients receiving chemotherapy; (iii) maintaining or stimulatinghematological function in patients in need thereof, including cancerpatients; (iv) maintaining or stimulating erythropoietin function orsynthesis in patients in need thereof, including cancer patients; (v)mitigating or preventing anemia in patients in need thereof, includingcancer patients; (vi) maintaining or stimulating pluripotent,multipotent, and unipotent normal stem cell function or synthesis inpatients in need thereof, including cancer patients; (vii) promoting thearrest or retardation of tumor progression in those cancer patientsreceiving chemotherapy; and (viii) increasing patient survival and/ordelaying tumor progression while maintaining or improving the quality oflife in cancer patients receiving chemotherapy.

A. Summary of the Results of the U.S. Phase II NSCLC Clinical Trial

Data was recently unblinded from a United States (U.S.) multicenterPhase II clinical trial of the Formula (I) compound Tavocept™ known asBNP7787, disodium 2,2′-dithio-bis-ethane sulfonate, and dimesna) andinvolving patients with advanced, Stage IIIB/IV, non-small cell lungcarcinoma (NSCLC), including the adenocarcinoma sub-type, who receivedthe chemotherapeutic drugs docetaxel and cisplatin (for purposes of thisdocument referred to as the “U.S. Phase II NSCLC Clinical Trial”).

The U.S. Phase II NSCLC Clinical Trial disclosed in the presentinvention was used to ascertain the effect of a dose-denseadministration of docetaxel and cisplatin every two weeks withconcomitant administration of pegfilgrastim and darbepoetin alfa withand without administration of Tavocept™ (also referred to in theliterature as disodium 2,2′-dithio-bis-ethane sulfonate, dimesna, orBNP7787) in patients with advanced stage (IIIB/IV) non-small cell lungcarcinoma (NSCLC), including the adenocarcinoma sub-type. Whether or notTavocept™ would affect the efficacy of the dose-densedocetaxel/cisplatin combination therapy was also evaluated based on theresponse rate, aggravation-free survival period, and total survivalperiod. In order to make all these evaluations, in the Tavocept™ arm ofthe U.S. Phase II NSCLC Clinical Trial, docetaxel administration (75mg/m²; i.v. administration over a period of 1 hour on day one of thechemotherapy cycle) was immediately followed by the administration ofTavocept™ (approximately 40 grams; i.v. administration over a period of30 minutes). The Tavocept™ administration was then immediately followedby the administration of cisplatin (75 mg/m²; i.v. administration over aperiod of 1 hour) with adequate hydration. Darbepoetin alfa (200 μg;subcutaneous administration) was administered on day one of thechemotherapy cycle and pegfilgrastim (6 mg subcutaneous administration)was administered on day two of the chemotherapy cycle if the patient'shemoglobin levels were ≦11 g/dL. The aforementioned chemotherapy cyclewas repeated every two weeks, for up to a total of six cycles. Theother, non-Tavocept™ administration arm of the study was identical tothe previously discussed Tavocept™ arm, with the exception that thedocetaxel administration was immediately followed by cisplatinadministration without an intermediate administration of Tavocept™. Inaddition, the incidence and severity of Grade 3 and Grade 4 adverseevents were compared between patients in the Tavocept™ and non-Tavocept™administration arms of the U.S. Phase II NSCLC Clinical Trial using theNational Cancer Institute-Common Toxicity Criteria (NCI-CTC)questionnaire.

B. Summary of the Results of the U.S. Phase II NSCLC Clinical Trial

The U.S. Phase II NSCLC Clinical Trial data demonstratedmedically-important reductions in the chemotherapy-induced side effectsof dehydration, nausea, vomiting, and a dramatic reduction inhypomagnesaemia.

The aforementioned clinical trial also provided a number of unexpectedphysiological results which have, heretofore, been unreported in anyprevious scientific or clinical studies, with the exception of the JapanPhase III Clinical Trial. Similar to the results obtained in the JapanPhase III Clinical Trial, the U.S. Phase II NSCLC Clinical Trialdemonstrated increased survival times for patients with advancednon-small cell lung cancer (NSCLC), including the adenocarcinomasub-type, receiving Tavocept™ and chemotherapy. A marked increase insurvival time was also observed in those patients with theadenocarcinoma non-small cell lung carcinoma (NSCLC) sub-type receivingTavocept™ and chemotherapy. In addition, the unexpected and novelresults for the Japan Phase III Clinical Trial and/or the U.S. Phase IINSCLC Clinical Trial included, but were not limited to: (i) potentiationof the cytotoxic or apoptotic activities of chemotherapeutic agents inpatients with non-small cell lung carcinoma, including theadenocarcinoma sub-type, receiving Tavocept™ and chemotherapy and (ii)increasing patient survival and/or delaying tumor progression whileconcomitantly maintaining or improving the quality of life in patientswith non-small cell lung carcinoma, including the adenocarcinomasub-type, receiving Tavocept™ and chemotherapy due to a reduction inseveral chemotherapy-induced physiological side effects. It should benoted that in the U.S. Phase II NSCLC Clinical Trial, unlike the JapanPhase III Clinical Trial, the maintenance or stimulation ofhematological function (e.g., an increase in hemoglobin, hematocrit, anderythrocyte levels), in patients with non-small cell lung carcinoma,including adenocarcinoma, receiving Tavocept™ and chemotherapy was notmeasured due to the fact that patients with hemoglobin levels ≦11 g/dL,received darbepoetin alfa (200 μg) and pegfilgrastim (6 mg) on day 1 andday 2 of the patient's chemotherapy cycle, respectively.

FIG. 12 illustrates, in graphical form, the median patient survival(i.e., time to death in months) in the U.S. Phase II NSCLC ClinicalTrial, in patient populations diagnosed with non-small cell lungcarcinoma, including the adenocarcinoma sub-type, receiving chemotherapywith either Tavocept™ (BNP7787) or no Tavocept™ treatment. The resultsindicate a 0.92 month increase in patient survival in the Tavocept™ armof the study (11.66 months) versus the non-Tavocept™ arm (10.74 months)measured with a 95% confidence limit. The hazard ratio was 0.750.

FIG. 13 illustrates, in tabular form, patient overall survival (OS) andpatient progression-free survival (PFS) in the U.S. Phase II NSCLCClinical Trial, in patient populations diagnosed with non-small celllung carcinoma, including the adenocarcinoma sub-type, receivingchemotherapy with either Tavocept™ (BNP7787) or no Tavocept™ treatment.The results indicate a 9.5% increase in patient progression-freesurvival (PFS) in the Tavocept™ arm of the study (18.7%) versus thenon-Tavocept™ arm (9.25%) and an 11.2% increase in overall patientone-year survival (OS) rates in the Tavocept™ arm (50.7%) verses thenon-Tavocept™ arm (39.5%), both values measured with a 95% confidenceinterval.

FIG. 14 illustrates, in graphical form, the median patient survival(i.e., time to death in months) in the U.S. Phase II NSCLC Phase IIClinical Trial, in patient populations diagnosed with adenocarcinomareceiving chemotherapy with either Tavocept™ (BNP7787) or no Tavocept™treatment. The results indicate a 6.54 month increase in patientsurvival in the Tavocept™ arm of the study (15.64 months) versus thenon-Tavocept™ arm (9.10 months). This value was measured with a 95%confidence limit. This represents a 40% reduction in the patientmortality rate. In addition, it should be noted that there were overdouble the number of patients in the Tavocept™ arm of the study (11patients) verses the non-Tavocept™ arm (5 patients). The hazard ratiowas 0.601.

FIG. 15 illustrates, in tabular form, the number of patientsexperiencing Grade 3 and Grade 4 treatment-related adverse events in theU.S. Phase II NSCLC Phase II Clinical Trial, in patient populationsdiagnosed with non-small cell lung carcinoma, including theadenocarcinoma sub-type, receiving chemotherapy with either Tavocept™(BNP7787) or no Tavocept™ treatment. The results indicate a 50%reduction in dehydration, a 38.5% reduction in nausea, a 71.5% reductionin vomiting, and a 100% reduction in hypomagnesaemia in the patients inthe Tavocept™ arm of the study versus the non-Tavocept™ arm.

In summation, the Applicant believes the experimental and clinical dataobtained from the Japan Phase III Clinical Trial and the U.S. Phase IINSCLC Clinical Trial, discussed above, supports the ability of Tavocept™to cause a marked increase in the survival time of patients withnon-small cell lung carcinoma (NSCLC), and especially in patients withthe adenocarcinoma NSCLC sub-type. It is important to note that thepatient populations in the U.S. Phase II NSCLC Clinical Trial and JapanPhase III Clinical Trial taken together represent a diverse sampling ofpatients having different ethnicities. Additional experimental andclinical evaluation will lend continued support for the ability ofTavocept™ to increase the survival time of patients with cancer, whereinthe cancer either: (i) overexpresses thioredoxin or glutaredoxin and/or(ii) exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapeutic agent or agents used to treat saidpatient with cancer.

All patents, publications, scientific articles, web sites, and the like,as well as other documents and materials referenced or mentioned hereinare indicative of the levels of skill of those skilled in the art towhich the invention pertains, and each such referenced document andmaterial is hereby incorporated by reference to the same extent as if ithad been incorporated by reference in its entirety individually or setforth herein in its entirety. Applicant reserves the right to physicallyincorporate into this specification any and all materials andinformation from any such patents, publications, scientific articles,web sites, electronically available information, and other referencedmaterials or documents.

The written description portion of this patent includes all claims.Furthermore, all claims, including all original claims as well as allclaims from any and all priority documents, are hereby incorporated byreference in their entirety into the written description portion of thespecification, and Applicant reserves the right to physicallyincorporate into the written description or any other portion of theapplication, any and all such claims. Thus, for example, under nocircumstances may the patent be interpreted as allegedly not providing awritten description for a claim on the assertion that the precisewording of the claim is not set forth in haec verba in the writtendescription portion of the patent.

The claims will be interpreted according to law. However, andnotwithstanding the alleged or perceived ease or difficulty ofinterpreting any claim or portion thereof, under no circumstances mayany adjustment or amendment of a claim or any portion thereof duringprosecution of the application or applications leading to this patent beinterpreted as having forfeited any right to any and all equivalentsthereof that do not form a part of the prior art.

All of the features disclosed in this specification may be combined inany combination. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Thus,from the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention. Other aspects, advantages,and modifications are within the scope of the following claims and thepresent invention is not limited except as by the appended claims.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,the terms “comprising”, “including”, “containing”, etc. are to be readexpansively and without limitation. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and they are not necessarily restricted to the ordersof steps indicated herein or in the claims.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by various embodiments and/or preferredembodiments and optional features, any and all modifications andvariations of the concepts herein disclosed that may be resorted to bythose skilled in the art are considered to be within the scope of thisinvention as defined by the appended claims.

The present invention has been described broadly and generically herein.Each of the narrower species and subgeneric groupings falling within thegeneric disclosure also form part of the invention. This includes thegeneric description of the invention with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

It is also to be understood that as used herein and in the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise, the term “X and/or Y”means “X” or “Y” or both “X” and “Y”. The letter “s” following a noundesignates both the plural and singular forms of that noun. In addition,where features or aspects of the invention are described in terms ofMarkush groups, it is intended, and those skilled in the art willrecognize, that the invention embraces and is also thereby described interms of any individual member and any subgroup of members of theMarkush group, and Applicant reserves the right to revise theapplication or claims to refer specifically to any individual member orany subgroup of members of the Markush group.

Other embodiments are within the following claims. The patent may not beinterpreted to be limited to the specific examples or embodiments ormethods specifically and/or expressly disclosed herein. Under nocircumstances may the patent be interpreted to be limited by anystatement made by any Examiner or any other official or employee of thePatent and Trademark Office unless such statement is specifically andwithout qualification or reservation expressly adopted in a responsivewriting by Applicants.

What is claimed is: 1) A method for increasing survival time in apatient with cancer, wherein said cancer, either: (i) overexpressesthioredoxin or glutaredoxin and/or (ii) exhibits evidence ofthioredoxin-mediated or glutaredoxin-mediated resistance to thechemotherapy agent or agents used to treat said patient with cancer;wherein said method comprises the administration of amedically-sufficient dose of a Formula (I) compound to said patient withcancer either prior to, concomitantly with, or subsequent to theadministration of a chemotherapy agent or agents whose cytotoxic orcytostatic activity is adversely affected by either: (i) theoverexpression of thioredoxin or glutaredoxin and/or (ii)thioredoxin-mediated or glutaredoxin-mediated treatment resistance, andwherein administration of said Formula (I) compound occurs prior to,concomitantly with, or subsequent to the administration of one or moreenzymes, proteins, peptides, or polyclonal and/or monoclonal antibodiesthat are also being administered to treat said cancer. 2) The method ofclaim 1, wherein the cancer is selected from the group consisting of:lung cancer, colorectal cancer, gastric cancer, esophageal cancer,ovarian cancer, cancer of the biliary tract, gallbladder cancer,cervical cancer, breast cancer, endometrial cancer, vaginal cancer,prostate cancer, uterine cancer, hepatic cancer, pancreatic cancer, andadenocarcinoma. 3) A method of increasing survival time in a patientwith non-small cell lung carcinoma, wherein the non-small lungcarcinoma, either: (i) overexpresses thioredoxin or glutaredoxin and/or(ii) exhibits evidence of thioredoxin-mediated or glutaredoxin-mediatedresistance to the chemotherapy agent or agents used to treat saidpatient with non-small cell lung carcinoma; wherein said methodcomprises the administration of a medically-sufficient dose of a Formula(I) compound to said patient either prior to, concomitantly with, orsubsequent to the administration of a chemotherapy agent or agents whosecytotoxic or cytostatic activity is adversely affected by either: (i)the overexpression of thioredoxin or glutaredoxin and/or (ii)thioredoxin-mediated or glutaredoxin-mediated treatment resistance, andwherein administration of said Formula (I) compound occurs prior to,concomitantly with, or subsequent to the administration of one or moreenzymes, proteins, peptides, or polyclonal and/or monoclonal antibodiesthat are also being administered to treat said cancer. 4) A method ofincreasing survival time in a patient with adenocarcinoma, wherein theadenocarcinoma, either: (i) overexpresses thioredoxin or glutaredoxinand/or (ii) exhibits evidence of thioredoxin-mediated orglutaredoxin-mediated resistance to the chemotherapy agent or agentsused to treat said patient with adenocarcinoma; wherein said methodcomprises the administration of a medically-sufficient dose of a Formula(I) compound to said patient either prior to, concomitantly with, orsubsequent to the administration of a chemotherapy agent or agents whosecytotoxic or cytostatic activity is adversely affected by either: (i)the overexpression of thioredoxin or glutaredoxin and/or (ii)thioredoxin-mediated or glutaredoxin-mediated treatment resistance, andwherein administration of said Formula (I) compound occurs prior to,concomitantly with, or subsequent to the administration of one or moreenzymes, proteins, peptides, or polyclonal and/or monoclonal antibodiesthat are also being administered to treat said cancer. 5) The method ofclaim 1, claim 3, or claim 4, wherein said Formula (I) compound has thestructural formula:X—S—S—R₁-R₂: wherein; R₁ is a lower alkylene, wherein R₁ is optionallysubstituted by a member of the group consisting of: lower alkyl, aryl,hydroxy, alkoxy, aryloxy, mercapto, alkylthio or arylthio, for acorresponding hydrogen atom, or

R₂ and R₄ is sulfonate or phosphonate; R₅ is hydrogen, hydroxy, orsulfhydryl; m is 0, 1, 2, 3, 4, 5, or 6; and X is a sulfur-containingamino acid or a peptide consisting of from 2-10 amino acids; or whereinX is a member of the group consisting of: lower thioalkyl (lowermercapto alkyl), lower alkylsulfonate, lower alkylphosphonate, loweralkenylsulfonate, lower alkyl, lower alkenyl, lower alkynyl, aryl,alkoxy, aryloxy, mercapto, alkylthio or hydroxy for a correspondinghydrogen atom; and pharmaceutically-acceptable salts, prodrugs, analogs,conjugates, hydrates, solvates, polymorphs, stereoisomers (includingdiastereoisomers and enantiomers) and tautomers thereof. 6) The methodof claim 5, wherein said Formula (I) compound is selected from the groupconsisting of: a disodium salt, a monosodium salt, a sodium potassiumsalt, a dipotassium salt, a monopotassium salt, a calcium salt, amagnesium salt, an ammonium salt, or a manganese salt. 7) The method ofclaim 5, wherein said Formula (I) compound is a disodium salt. 8) Themethod of claim 1, claim 3, or claim 4, wherein said Formula (I)compound is disodium 2,2′-dithio-bis-ethane sulfonate. 9) The method ofclaim 1, claim 3, or claim 4, wherein said Formula (I) compoundcomprises 2-mercapto-ethane sulfonate or 2-mercapto-ethane sulfonateconjugated as a disulfide with a substituent group selected from thegroup consisting of: -Cys, -Homocysteine, -Cys-Gly, -Cys-Glu,-Homocysteine, -Homocysteine-Gly, -Homocysteine-Glu, -Cys-Glu,

wherein R₁ and R₂ are any L- or D-amino acids; andpharmaceutically-acceptable salts thereof. 10) The method of claim 1,claim 3, or claim 4, wherein said chemotherapy agent or agents areselected from the group consisting of: fluoropyrimidines; pyrimidinenucleosides; purine nucleosides; anti-folates, platinum agents;anthracyclines/anthracenediones; epipodophyllotoxins; camptothecins;hormones; hormonal complexes; antihormonals; enzymes, proteins, peptidesand polyclonal and/or monoclonal antibodies; vinca alkaloids; taxanes;epothilones; antimicrotubule agents; alkylating agents; antimetabolites;topoisomerase inhibitors; aziridine-containing compounds; antivirals;and various other cytotoxic and cytostatic agents. 11) The method ofclaim 1, claim 3, or claim 4, wherein said chemotherapy agent or agentsare selected from the group consisting of: cisplatin, carboplatin,oxaliplatin, satraplatin, picoplatin, tetraplatin, platinum-DACH, andanalogs and derivatives thereof. 12) The method of claim 1, claim 3, orclaim 4, wherein said chemotherapy agent or agents are selected from thegroup consisting of: docetaxel, paclitaxel, polyglutamylated forms ofpaclitaxel, liposomal paclitaxel, and analogs and derivatives thereof.13) The method of claim 1, claim 3, or claim 4, wherein the chemotherapyagents are docetaxel and cisplatin. 14) The method of claim 1, claim 3,or claim 4, wherein the chemotherapy agents are paclitaxel andcisplatin. 15) The method of claim 1, claim 3, or claim 4, wherein saidenzymes, proteins, peptides, and polyclonal and/or monoclonal antibodiesare selected from the group consisting of: asparaginase, cetuximab,erlotinib, bevacizumab, rituximab, gefitinib, trastuzumab, interleukins,interferons, leuprolide, and pegasparaginase. 16) The method of claim 1,claim 3, or claim 4, wherein said monoclonal antibodies are cetuximab orbevacizumab. 17) A kit comprising a Formula (I) compound foradministration, and instructions for administering said Formula (I)compound to a patient with cancer in an amount sufficient to cause anincrease in the survival time of said patient with cancer who isreceiving a chemotherapy agent or agents whose cytotoxic or cytostaticactivity is adversely affected by either: (i) the overexpression ofthioredoxin or glutaredoxin and/or (ii) thioredoxin-mediated orglutaredoxin-mediated treatment resistance, and wherein administrationof said Formula (I) compound occurs prior to, concomitantly with, orsubsequent to the administration of one or more enzymes, proteins,peptides, or polyclonal and/or monoclonal antibodies that are also beingadministered to treat said cancer. 18) The kit of claim 17, wherein thecancer is selected from the group consisting of any cancer which either:(i) overexpresses thioredoxin or glutaredoxin and/or (ii) exhibitsevidence of thioredoxin-mediated or glutaredoxin-mediated resistance tothe chemotherapy agent or agents being used to treat said cancer. 19)The kit of claim 17, wherein the cancer is selected from the groupconsisting of: lung cancer, colorectal cancer, gastric cancer,esophageal cancer, ovarian cancer, cancer of the biliary tract,gallbladder cancer, cervical cancer, breast cancer, endometrial cancer,vaginal cancer, prostate cancer, uterine cancer, hepatic cancer,pancreatic cancer, and adenocarcinoma. 20) A kit comprising a Formula(I) compound for administration, and instructions for administering saidFormula (I) compound to a patient with non-small cell lung carcinoma inan amount sufficient to cause an increase in the survival time of saidpatient who is receiving a chemotherapy agent or agents whose cytotoxicor cytostatic activity is adversely affected by either: (i) theoverexpression of thioredoxin or glutaredoxin and/or (ii)thioredoxin-mediated or glutaredoxin-mediated treatment resistance, andwherein administration of said Formula (I) compound occurs prior to,concomitantly with, or subsequent to the administration of one or moreenzymes, proteins, peptides, or polyclonal and/or monoclonal antibodiesthat are also being administered to treat said cancer. 21) A kitcomprising a Formula (I) compound for administration, and instructionsfor administering said Formula (I) compound to a patient withadenocarcinoma in an amount sufficient to cause an increase in thesurvival time of said patient who is receiving a chemotherapy agent oragents whose cytotoxic or cytostatic activity is adversely affected byeither: (i) the overexpression of thioredoxin or glutaredoxin and/or(ii) thioredoxin-mediated or glutaredoxin-mediated treatment resistance,and wherein administration of said Formula (I) compound occurs prior to,concomitantly with, or subsequent to the administration of one or moreenzymes, proteins, peptides, or polyclonal and/or monoclonal antibodiesthat are also being administered to treat said cancer. 22) The kit ofclaim 17, claim 20, or claim 21, wherein said Formula (I) compound hasthe structural formula:X—S—S—R—R₂: wherein; R₁ is a lower alkylene, wherein R₁ is optionallysubstituted by a member of the group consisting of lower alkyl, aryl,hydroxy, alkoxy, aryloxy, mercapto, alkylthio or arylthio, for acorresponding hydrogen atom, or

R₂ and R₄ is sulfonate or phosphonate; R₅ is hydrogen, hydroxy, orsulfhydryl; m is 0, 1, 2, 3, 4, 5, or 6; and X is a sulfur-containingamino acid or a peptide consisting of from 2-10 amino acids; or whereinX is a member of the group consisting of: lower thioalkyl (lowermercapto alkyl), lower alkylsulfonate, lower alkylphosphonate, loweralkenylsulfonate, lower alkyl, lower alkenyl, lower alkynyl, aryl,alkoxy, aryloxy, mercapto, alkylthio or hydroxy for a correspondinghydrogen atom; and pharmaceutically-acceptable salts, prodrugs, analogs,conjugates, hydrates, solvates, polymorphs, stereoisomers (includingdiastereoisomers and enantiomers) and tautomers thereof. 23) The kit ofclaim 22, wherein said Formula (I) compound is selected from the groupconsisting of: a disodium salt, a monosodium salt, a sodium potassiumsalt, a dipotassium salt, a monopotassium salt, a calcium salt, amagnesium salt, an ammonium salt, or a manganese salt. 24) The kit ofclaim 22, wherein said Formula (I) compound is a disodium salt. 25) Thekit of claim 17, claim 20, or claim 21, wherein said Formula (I)compound is disodium 2,2′-dithio-bis-ethane sulfonate. 26) The kit ofclaim 17, claim 20, or claim 21, wherein said Formula (I) compoundcomprises 2-mercapto-ethane sulfonate or 2-mercapto-ethane sulfonateconjugated as a disulfide with a substituent group selected from thegroup consisting of: -Cys, -Homocysteine, -Cys-Gly, -Cys-Glu,-Homocysteine, -Homocysteine-Gly, -Homocysteine-Glu, -Cys-Glu,

wherein R₁ and R₂ are any L- or D-amino acids; andpharmaceutically-acceptable salts thereof. 27) The kit of claim 17,claim 20, or claim 21, wherein said chemotherapy agent or agents areselected from the group consisting of: fluoropyrimidines; pyrimidinenucleosides; purine nucleosides; anti-folates, platinum agents;anthracyclines/anthracenediones; epipodophyllotoxins; camptothecins;hormones; hormonal complexes; antihormonals; enzymes, proteins, peptidesand polyclonal and/or monoclonal antibodies; vinca alkaloids; taxanes;epothilones; antimicrotubule agents; alkylating agents; antimetabolites;topoisomerase inhibitors; aziridine-containing compounds; antivirals;and various other cytotoxic and cytostatic agents. 28) The kit of claim17, claim 20, or claim 21, wherein said chemotherapy agent or agents areselected from the group consisting of: cisplatin, carboplatin,oxaliplatin, satraplatin, picoplatin, tetraplatin, platinum-DACH, andanalogs and derivatives thereof. 29) The kit of claim 17, claim 20, orclaim 21, wherein said chemotherapy agent or agents are selected fromthe group consisting of: docetaxel, paclitaxel, polyglutamylated formsof paclitaxel, liposomal paclitaxel, and analogs and derivativesthereof. 30) The kit of claim 17, claim 20, or claim 21, wherein thechemotherapy agents are docetaxel and cisplatin. 31) The method of claim17, claim 20, or claim 21, wherein the chemotherapy agents arepaclitaxel and cisplatin. 32) The kit of claim 17, claim 20, or claim21, wherein said enzymes, proteins, peptides, and polyclonal and/ormonoclonal antibodies are selected from the group consisting of:asparaginase, cetuximab, erlotinib, bevacizumab, rituximab, gefitinib,trastuzumab, interleukins, interferons, leuprolide, and pegasparaginase.33) The kit of claim 17, claim 20, or claim 21, wherein said monoclonalantibodies are cetuximab or bevacizumab. 34) A method for increasingpatient survival time and/or delaying tumor progression in a patientsuffering from cancer treated with a taxane and/or platinum chemotherapyagent or agents, wherein said method is comprised of the administrationof a Formula (I) compound to said patient, and wherein theadministration of said Formula (I) compound occurs prior to,concomitantly with or subsequent to the administration of one or moreenzymes, proteins, peptides, or polyclonal and/or monoclonal antibodiesthat are also being used to treat said cancer, and wherein said Formula(I) compound, said taxane and/or platinum chemotherapy agent or agents,and said one or more enzymes, proteins, peptides, or polyclonal and/ormonoclonal antibodies are all administered to the patient in medicallysufficient dosages. 35) The method of claim 34, wherein the cancer isselected from the group consisting of: lung cancer, colorectal cancer,gastric cancer, esophageal cancer, ovarian cancer, cancer of the biliarytract, gallbladder cancer, cervical cancer, breast cancer, endometrialcancer, vaginal cancer, prostate cancer, uterine cancer, hepatic cancer,pancreatic cancer, and adenocarcinoma. 36) A method for increasingpatient survival time and/or delaying tumor progression in a patientsuffering from non-small cell lung carcinoma treated with a taxaneand/or platinum chemotherapy agent or agents, wherein said method iscomprised of the administration of a Formula (I) compound to saidpatient wherein the administration of said Formula (I) compound occursprior to, concomitantly with or subsequent to the administration of oneor more enzymes, proteins, peptides, or polyclonal and/or monoclonalantibodies that are also being used to treat said non-small cell lungcarcinoma, and wherein said Formula (I) compound, said taxane and/orplatinum chemotherapy agent or agents, and said one or more enzymes,proteins, peptides, or polyclonal and/or monoclonal antibodies are alladministered to the patient in medically sufficient dosages. 37) Amethod for increasing patient survival time and/or delaying tumorprogression in a patient suffering from adenocarcinoma who is treatedwith a taxane and/or platinum chemotherapy agent or agents, wherein saidmethod is comprised of the administration of a Formula (I) compound tosaid patient wherein the administration of said Formula (I) compoundoccurs prior to, concomitantly with or subsequent to the administrationof one or more enzymes, proteins, peptides, or polyclonal and/ormonoclonal antibodies that are also being used to treat saidadenocarcinoma, and wherein said Formula (I) compound, said taxaneand/or platinum chemotherapy agent or agents, and said one or moreenzymes, proteins, peptides, or polyclonal and/or monoclonal antibodiesare all administered to the patient in medically sufficient dosages. 38)The method of any one of claims 34-37, wherein said increase in patientsurvival time in said patient treated with a Formula (I) compound isexpected to be at least 30 days longer than the expected survival timeif said patient was not treated with a Formula (I) compound. 39) Amethod for potentiating the chemotherapeutic effects of a taxane and/orplatinum chemotherapy agent or agents used to treat a patient sufferingfrom cancer, wherein said method is comprised of the administration of aFormula (I) compound to said patient, and wherein the administration ofsaid Formula (I) compound occurs prior to, concomitantly with orsubsequent to the administration of one or more enzymes, proteins,peptides, or polyclonal and/or monoclonal antibodies that are also beingused to treat said cancer, and wherein said Formula (I) compound, saidtaxane and/or platinum chemotherapy agent or agents, and said one ormore enzymes, proteins, peptides, or polyclonal and/or monoclonalantibodies are all administered to the patient in medically sufficientdosages. 40) The method of claim 39, wherein the cancer is selected fromthe group consisting of: lung cancer, colorectal cancer, gastric cancer,esophageal cancer, ovarian cancer, cancer of the biliary tract,gallbladder cancer, cervical cancer, breast cancer, endometrial cancer,vaginal cancer, prostate cancer, uterine cancer, hepatic cancer,pancreatic cancer, and adenocarcinoma. 41) A method for potentiating thechemotherapeutic effects of a taxane and/or platinum chemotherapy agentor agents used to treat a patient suffering from non-small cell lungcarcinoma, wherein said method is comprised of the administration of aFormula (I) compound to said patient, and wherein the administration ofsaid Formula (I) compound occurs prior to, concomitantly with orsubsequent to the administration of one or more enzymes, proteins,peptides, or polyclonal and/or monoclonal antibodies that are also beingused to treat said non-small cell lung carcinoma, and wherein saidFormula (I) compound, said taxane and/or platinum chemotherapy agent oragents, and said one or more enzymes, proteins, peptides, or polyclonaland/or monoclonal antibodies are all administered to the patient inmedically sufficient dosages. 42) A method for potentiating thechemotherapeutic effects of a taxane and/or platinum chemotherapy agentor agents used to treat a patient suffering from adenocarcinoma, whereinsaid method is comprised of the administration of a Formula (I) compoundto said patient, and wherein the administration of said Formula (I)compound occurs prior to, concomitantly with or subsequent to theadministration of one or more enzymes, proteins, peptides, or polyclonaland/or monoclonal antibodies that are also being used to treat saidadenocarcinoma, and wherein said Formula (I) compound, said taxaneand/or platinum chemotherapy agent or agents, and said one or moreenzymes, proteins, peptides, or polyclonal and/or monoclonal antibodiesare all administered to the patient in medically sufficient dosages. 43)A method for promoting the arrest or retardation of tumor progression ina patient suffering from cancer who is treated with a taxane and/orplatinum chemotherapy agent or agents, wherein said method is comprisedof the administration of a Formula (I) compound to said patient, andwherein the administration of said Formula (I) compound occurs prior to,concomitantly with or subsequent to the administration of one or moreenzymes, proteins, peptides, or polyclonal and/or monoclonal antibodiesthat are also being used to treat said cancer, and wherein said Formula(I) compound, said taxane and/or platinum chemotherapy agent or agents,and said one or more enzymes, proteins, peptides, or polyclonal and/ormonoclonal antibodies are all administered to the patient in medicallysufficient dosages. 44) The method of claim 43, wherein the cancer isselected from the group consisting of lung cancer, colorectal cancer,gastric cancer, esophageal cancer, ovarian cancer, cancer of the biliarytract, gallbladder cancer, cervical cancer, breast cancer, endometrialcancer, vaginal cancer, prostate cancer, uterine cancer, hepatic cancer,pancreatic cancer, and adenocarcinoma. 45) A method for promoting thearrest or retardation of tumor progression in a patient suffering fromnon-small cell lung carcinoma who is treated with a taxane and/orplatinum chemotherapy agent or agents, wherein said method is comprisedof the administration of a Formula (I) compound and the administrationof said Formula (I) compound occurs prior to, concomitantly with orsubsequent to the administration of one or more enzymes, proteins,peptides, or polyclonal and/or monoclonal antibodies that are also beingused to treat said non-small cell lung carcinoma, and wherein saidFormula (I) compound, said taxane and/or platinum chemotherapy agent oragents, and said one or more enzymes, proteins, peptides, or polyclonaland/or monoclonal antibodies are all administered to the patient inmedically sufficient dosages. 46) A method for promoting the arrest orretardation of tumor progression in a patient suffering fromadenocarcinoma who is treated with a taxane and/or platinum chemotherapyagent or agents, wherein said method is comprised of the administrationof a Formula (I) compound to said patient, and wherein theadministration of said Formula (I) compound occurs prior to,concomitantly with or subsequent to the administration of one or moreenzymes, proteins, peptides, or polyclonal and/or monoclonal antibodiesthat are also being used to treat said adenocarcinoma, and wherein saidFormula (I) compound, said taxane and/or platinum chemotherapy agent oragents, and said one or more enzymes, proteins, peptides, or polyclonaland/or monoclonal antibodies are all administered to the patient inmedically sufficient dosages. 47) A method for increasing the survivaltime while concomitantly maintaining or increasing the quality of lifein a patient suffering from cancer who is treated with a taxane and/orplatinum chemotherapy agent or agents, wherein said method is comprisedof the administration of a Formula (I) compound to said patient, andwherein the administration of said Formula (I) compound occurs prior to,concomitantly with or subsequent to the administration of one or moreenzymes, proteins, peptides, or polyclonal and/or monoclonal antibodiesthat are also being used to treat said cancer, and wherein said Formula(I) compound, said taxane and/or platinum chemotherapy agent or agents,and said one or more enzymes, proteins, peptides, or polyclonal and/ormonoclonal antibodies are all administered to the patient in medicallysufficient dosages. 48) The method of claim 47, wherein the cancer isselected from the group consisting of lung cancer, colorectal cancer,gastric cancer, esophageal cancer, ovarian cancer, cancer of the biliarytract, gallbladder cancer, cervical cancer, breast cancer, endometrialcancer, vaginal cancer, prostate cancer, uterine cancer, hepatic cancer,pancreatic cancer, and adenocarcinoma. 49) A method for increasing thesurvival time while concomitantly maintaining or increasing the qualityof life in a patient suffering from non-small cell lung carcinoma who istreated with a taxane and/or platinum chemotherapy agent or agents,wherein said method is comprised of the administration of a Formula (I)compound to said patient, and wherein the administration of said Formula(I) compound occurs prior to, concomitantly with or subsequent to theadministration of one or more enzymes, proteins, peptides, or polyclonaland/or monoclonal antibodies that are also being used to treat saidnon-small cell lung carcinoma, and wherein said Formula (I) compound,said taxane and/or platinum chemotherapy agent or agents, and said oneor more enzymes, proteins, peptides, or polyclonal and/or monoclonalantibodies are all administered to the patient in medically sufficientdosages. 50) A method for increasing the survival time whileconcomitantly maintaining or increasing the quality of life in a patientsuffering from adenocarcinoma who is treated with a taxane and/orplatinum chemotherapy agent or agents, wherein said method is comprisedof the administration of a Formula (I) compound to said patient, andwherein the administration of said Formula (I) compound occurs prior to,concomitantly with or subsequent to the administration of one or moreenzymes, proteins, peptides, or polyclonal and/or monoclonal antibodiesthat are also being used to treat said adenocarcinoma, and wherein saidFormula (I) compound, said taxane and/or platinum chemotherapy agent oragents, and said one or more enzymes, proteins, peptides, or polyclonaland/or monoclonal antibodies are all administered to the patient inmedically sufficient dosages. 51) A method for increasing the survivaltime while concomitantly affecting hematological function in a patientsuffering from cancer who is treated with a taxane and/or platinumchemotherapy agent or agents, wherein said method is comprised of theadministration of a Formula (I) compound to said patient, the effect onhematological function is selected from the group consisting of: (i)maintaining or stimulating hematological function, (ii) maintaining orstimulating erythropoietin function or synthesis, (iii) mitigating orpreventing anemia, and (iv) maintaining or stimulating pluripotent,multipotent, and unipotent normal stem cell function, and theadministration of said Formula (I) compound to said patient occurs priorto, concomitantly with or subsequent to the administration of one ormore enzymes, proteins, peptides, or polyclonal and/or monoclonalantibodies that are also being used to treat said cancer, and whereinsaid Formula (I) compound, said taxane and/or platinum chemotherapyagent or agents, and said one or more enzymes, proteins, peptides, orpolyclonal and/or monoclonal antibodies are all administered to thepatient in medically sufficient dosages. 52) The method of any one ofclaims 34-51, wherein said Formula (I) compound has the structuralformula:X—S—S—R₁-R₂: wherein; R₁ is a lower alkylene, wherein R₁ is optionallysubstituted by a member of the group consisting of: lower alkyl, aryl,hydroxy, alkoxy, aryloxy, mercapto, alkylthio or arylthio, for acorresponding hydrogen atom, or

R₂ and R₄ is sulfonate or phosphonate; R₅ is hydrogen, hydroxy, orsulthydryl; m is 0, 1, 2, 3, 4, 5, or 6; and X is a sulfur-containingamino acid or a peptide consisting of from 2-10 amino acids; or whereinX is a member of the group consisting of: lower thioalkyl (lowermercapto alkyl), lower alkylsulfonate, lower alkylphosphonate, loweralkenylsulfonate, lower alkyl, lower alkenyl, lower alkynyl, aryl,alkoxy, aryloxy, mercapto, alkylthio or hydroxy for a correspondinghydrogen atom; and pharmaceutically-acceptable salts, prodrugs, analogs,conjugates, hydrates, solvates, polymorphs, stereoisomers (includingdiastereoisomers and enantiomers) and tautomers thereof. 53) The methodof claim 52, wherein said Formula (I) compound is selected from thegroup consisting of: a disodium salt, a monosodium salt, a sodiumpotassium salt, a dipotassium salt, a monopotassium salt, a calciumsalt, a magnesium salt, an ammonium salt, or a manganese salt. 54) Themethod of claim 52, wherein said Formula (I) compound is a disodiumsalt. 55) The method of claim 52, wherein said Formula (I) compound isdisodium 2,2′-dithio-bis-ethane sulfonate. 56) The method of any one ofclaims 34-51, wherein said Formula (I) compound comprises2-mercapto-ethane sulfonate or 2-mercapto-ethane sulfonate conjugated asa disulfide with a substituent group selected from the group consistingof: -Cys, -Homocysteine, -Cys-Gly, -Cys-Glu, -Homocysteine,-Homocysteine-Gly, -Homocysteine-Glu, -Cys-Glu,

wherein R₁ and R₂ are any L- or D-amino acids; andpharmaceutically-acceptable salts thereof. 57) The method of any one ofclaims 34-51, wherein said platinum chemotherapy agent or agents areselected from the group consisting of: cisplatin, carboplatin,oxaliplatin, satraplatin, picoplatin, tetraplatin, platinum-DACH, andanalogs and derivatives thereof. 58) The method of any one of claims34-51, wherein said taxane chemotherapy agent or agents are selectedfrom the group consisting of: docetaxel, paclitaxel, polyglutamylatedforms of paclitaxel, liposomal paclitaxel, and analogs and derivativesthereof. 59) The method of any one of claims 34-51, wherein the taxaneand/or platinum chemotherapy agents are docetaxel and/or cisplatin. 60)The method of any one of claims 34-51, wherein the taxane and/orplatinum chemotherapy agents are paclitaxel and/or cisplatin. 61) Themethod of any one of claims 34-51, wherein said enzymes, proteins,peptides, or polyclonal and/or monoclonal antibodies are selected fromthe group consisting of asparaginase, cetuximab, erlotinib, bevacizumab,rituximab, gefitinib, trastuzumab, interleukins, interferons,leuprolide, and pegasparaginase. 62) The method of any one of claims34-51, wherein said monoclonal antibodies are cetuximab or bevacizumab.